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+ | </style> | ||
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+ | <div> | ||
+ | <div class="box-dark"> | ||
+ | <h1 class="heading"> | ||
+ | R E S U L T S | ||
+ | <!-- Titel austauschen--> | ||
+ | </h1> | ||
+ | <hr class="line"> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg" class="logo" alt="Syntex Logo"> | ||
</div> | </div> | ||
<div style="margin-top: 10vh;"> | <div style="margin-top: 10vh;"> | ||
− | + | <section class="section"> | |
− | + | <h1 class="title"> </h1> | |
− | + | <p style="text-align: justify;"> | |
− | + | <!-- Text--> | |
− | + | The way to the results we demonstrate here was full of success and failure. Therefore, it was | |
− | + | necessary | |
− | + | to | |
− | + | compare and revise our theoretical plans with the practical work and the associated results. After | |
− | + | trying our | |
− | + | best to implement our plans, we would like to show you on this page that we have managed to realize | |
− | + | some | |
− | + | of | |
− | + | our | |
− | + | goals and are able to show some achievements for every sub-group. | |
− | + | </p> | |
− | + | </section> | |
− | + | <hr> | |
− | + | <section class="section grid"> | |
− | + | <!--pop up faengt an--> | |
− | + | <div class="sub" onclick="popup('strain_engineering')"> | |
− | + | <div class="sub-header"> | |
− | + | <h1> | |
− | + | <!-- Titel von Pop up in schwarzer Box--> | |
− | + | S T R A I N<br> | |
− | + | E N G I N E E R I N G | |
− | + | </h1> | |
− | + | <hr> | |
− | + | </div> | |
− | + | <div class="sub-content"> | |
− | + | <div> | |
− | + | By genetic modification of <i>S. elongatus</i> UTEX 2973 we succeeded the transformation of | |
− | + | plasmids in UTEX | |
− | + | 2973. | |
− | + | </div> | |
+ | </div> | ||
</div> | </div> | ||
− | + | <div id="strain_engineering" class="popup"> | |
− | + | <!-- nummer aendern --> | |
− | + | <div class="popup-container"> | |
− | + | <div class="popup-header"> | |
− | + | <h1 class="title">Strain engineering</h1> <!-- Titel im pop up--> | |
− | + | <button type="button" onclick="hide('strain_engineering')">X</button> | |
− | + | <!-- nummer aendern --> | |
− | + | </div> | |
− | + | <div class="popup-content" style="text-align: justify;"> | |
− | + | <section class="section"> | |
− | + | <!-- Inhalt Pop up start--> | |
− | + | <p> | |
− | + | <u>Natural competence</u> | |
− | + | ||
− | + | <p> | |
− | + | Reintroducing natural competence into our <i>S. elongatus</i> strain was an | |
− | + | important goal for us. | |
− | + | To | |
− | + | make sure | |
− | + | that | |
− | + | our <i>S. elongatus</i> UTEX 2973 actually holds the point mutation in the | |
− | + | <i>pilN</i> gene we | |
− | + | thought | |
− | + | it has, | |
− | + | we sequenced | |
− | + | this region - and the results showed the expected mutation | |
− | + | <figure Style="text-align:center"> | |
− | + | <img style="height: 50ex; width: 90ex" | |
− | + | src=https://static.igem.org/mediawiki/2019/e/ed/T--Marburg--Seq_UTEX-pilN-with-o_iGEM_1_650.png | |
− | + | alt="sequencing results"> | |
− | + | <figcaption> | |
− | + | Fig.1: Sequencing results showing the point mutation in the <i>pilN</i> | |
− | <br> | + | gene. |
− | + | </figcaption> | |
− | + | </figure> | |
− | + | <br> | |
− | + | </p> | |
− | + | <p> | |
− | + | As there were multiple methods at hand that we could use to get our strain | |
− | + | naturally | |
− | + | competent | |
− | + | again, | |
− | + | we tried | |
− | + | all those at hand, making sure we do everything we can to tackle this issue. | |
− | + | <br> | |
− | + | Although not our favorite method, we tried integrating an intact copy of the | |
− | + | <i>pilN</i> gene into | |
− | + | neutral site | |
− | + | II of | |
− | + | <i>S. elongatus</i> UTEX 2973 following the example of <a | |
− | + | href=https://doi.org/10.1016/j.ymben.2018.06.002> Li <i>et al.</i>, 2018 | |
− | + | </a> . | |
− | + | We received pSII-trc-pilN, | |
− | + | the same plasmid used by Li <i>et al.</i>, as a gift from Petra Wurmser from the | |
− | + | research group of Prof. | |
− | + | Kaldenhoff in Darmstadt and conjugated it into our strain via triparental | |
− | + | conjugation.</p> | |
− | + | <figure Style="text-align:center"> | |
− | + | <img style="height: 40ex; width: 50ex" | |
− | + | src=https://static.igem.org/mediawiki/2019/d/d4/T--Marburg--p_pilN-NSII.png | |
− | + | alt="map"> | |
− | + | <figcaption> | |
− | + | Fig. 2: Plasmidmap of pSII-trc-<i> pilN </i>. | |
− | + | </figcaption> | |
− | + | </figure> | |
− | + | ||
<br> | <br> | ||
− | After a whole week we could actually see growing colonies, though they were only from attempts with the | + | <p> |
− | + | Beforehand, Petra Wurmser told us that she was not able to successfully | |
+ | reproduce | ||
+ | the experiment, | ||
+ | motivating | ||
+ | us | ||
+ | to try it ourselves. | ||
+ | |||
+ | We made sure to follow the protocol in the above mentioned paper, transforming | ||
+ | pRL443 and pRL623 | ||
+ | into <i>E. | ||
+ | coli</i> | ||
+ | HB101 and pSII-trc-pilN into <i>E. coli</i> HB101. Overnight cultures of these | ||
+ | cells | ||
+ | were inoculated and grown to | ||
+ | OD600≈0.5. | ||
+ | After washing and incubating them together for half an hour, they were mixed | ||
+ | with a | ||
+ | exponentially | ||
+ | growing | ||
+ | <i>S. elongatus</i> culture and incubated for 30 minutes again. Thereafter the | ||
+ | mixture was blotted | ||
+ | on | ||
+ | sterile filters and | ||
+ | incubated on BG11 plates for 24h before being transferred onto BG11 plates | ||
+ | containing kanamycin | ||
+ | <a href=https://doi.org/10.1016/j.ymben.2018.06.002> (Li <i>et al.</i>, | ||
+ | 2018)</a>. | ||
+ | It is important to add | ||
+ | that we also went with another approach for conjugation: Martina Carrillo | ||
+ | Camacho | ||
+ | from the working | ||
+ | group of Prof. Dr. Tobias Erb provided us with the pRK2013 plasmid, mentioning | ||
+ | that | ||
+ | she uses it | ||
+ | for conjugation. So we transformed the plasmid into <i> E. coli </i> DH5ɑ and | ||
+ | pSII-trc-pilN into | ||
+ | HB101, performing the | ||
+ | same | ||
+ | procedure with them as stated above. | ||
+ | <br> | ||
+ | After a whole week we could actually see growing colonies, though they were only | ||
+ | from attempts | ||
+ | with the | ||
+ | pRK2013 plasmid. </p> | ||
<figure Style="text-align:center"> | <figure Style="text-align:center"> | ||
<img style="height: 50ex; width: 60ex" | <img style="height: 50ex; width: 60ex" | ||
− | src=https://static.igem.org/mediawiki/2019/e/e5/T--Marburg--Trafo-pilN-NSII-UDAR.jpg alt="map"> | + | src=https://static.igem.org/mediawiki/2019/e/e5/T--Marburg--Trafo-pilN-NSII-UDAR.jpg |
+ | alt="map"> | ||
<figcaption> | <figcaption> | ||
− | Fig.3 : Pictures of | + | Fig.3 : Pictures of agarplates with colonies received from the triparental |
− | + | conjugation with | |
+ | the | ||
+ | helperstrain pRK2013 and HB101, haboring the pSII-trc-<i>pilN</i> vector. | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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<br> | <br> | ||
− | In hope of better results we decided to try and revert the point mutation in the <i> pilN </i> gene with | + | <p> |
− | + | We were still excited, directly starting liquid cultures and | |
− | + | running | |
− | + | colony PCRs in hope to find our desired result. Although trying a wide variety | |
− | + | of | |
− | + | primer | |
− | + | combinations, | |
− | + | we were | |
− | + | not able to find any successful integrations, but as the strain was growing on | |
− | + | kanamycin and | |
− | + | colony PCR | |
− | + | does not | |
+ | always work correctly, we wanted to make sure and tried an actual | ||
+ | transformation: A | ||
+ | YFP construct | ||
+ | was | ||
+ | transformed | ||
+ | into our seemingly competent strain, allowing for easy selection afterwards. | ||
+ | Disappointingly we could not transform the strain. This was in accordance to the | ||
+ | results of | ||
+ | Petra | ||
+ | Wurmser, who | ||
+ | was also not able to reintroduce natural competence into <i>S. elongatus</i> | ||
+ | UTEX | ||
+ | 2973 through | ||
+ | this | ||
+ | method. | ||
+ | <br> | ||
+ | In hope of better results we decided to try and revert the point mutation in the | ||
+ | <i> | ||
+ | pilN </i> | ||
+ | gene with | ||
+ | a CRISPR/Cas12a system.While still working on the system itself as described in | ||
+ | the | ||
+ | <ahref="https://2019.igem.org/Team:Marburg/Design#strain_engineering" | ||
+ | target="_blank"> design section</a>, we used | ||
+ | the pSL2680 plasmid <a href=https://www.nature.com/articles/srep39681 | ||
+ | target="_blank"> (Ungerer and Pakrasi, 2016) | ||
+ | </a> to engineer our strain. We followed their protocol <a | ||
+ | href=https://www.addgene.org/85581/ target="_blank">(available here on | ||
+ | Addgene)</a> to construct a guiding | ||
+ | crRNA, leading the Cas12a enzyme to the mutated <i> | ||
+ | pilN </i> gene and cloned it together with the enzyme and a repair | ||
+ | template | ||
+ | to revert the | ||
+ | point | ||
+ | mutation. Sequencing results of all parts needed were correct, so the final | ||
+ | construct could be | ||
+ | transformed into our cyanobacteria. | ||
+ | In this approach triparental conjugation was performed slightly differently, | ||
+ | as | ||
+ | we wanted to | ||
+ | exactly | ||
+ | follow the provided protocol: | ||
+ | In contrary to the other method, a HB101 strain harboring pRL623 was again | ||
+ | transformed with | ||
+ | the | ||
+ | plasmid | ||
+ | of interest, in this case the fully assembled pSL2680 and the other HB101 | ||
+ | strain | ||
+ | just | ||
+ | contained | ||
+ | pRL443. | ||
+ | Again, both strains were mixed together and then inoculated before being | ||
+ | blotted | ||
+ | on filters on | ||
+ | BG11 | ||
+ | plates. This time, after only 6h the filters were transferred on BG11 plates | ||
+ | with kanamycin. | ||
+ | During our first run a few colonies were visible after just three days, but | ||
+ | getting cultures | ||
+ | to | ||
+ | grow | ||
+ | in | ||
+ | liquid media did not work as expected and the colonies did not look as | ||
+ | healthy | ||
+ | as hoped. | ||
+ | As we have had this kind of issue several times during our project and saw | ||
+ | that | ||
+ | in literature | ||
+ | the | ||
+ | colonies growing on the plates looked differently, we decided to reach out | ||
+ | to | ||
+ | experienced | ||
+ | researchers | ||
+ | working with the same <i>Synechococcus elongatus</i> strain. We contacted <a | ||
+ | href="https://2019.igem.org/Team:Marburg/Human_Practices#james_golden" | ||
+ | target="_blank">Prof. Dr. James W. Golden</a> | ||
+ | from | ||
+ | the | ||
+ | University of California, San Diego to ask for advice and | ||
+ | were | ||
+ | able to | ||
+ | discover a crucial difference in the way we work with our strain: While in | ||
+ | our | ||
+ | own lab this is | ||
+ | not | ||
+ | commonly done, the researchers from the Golden lab put | ||
+ | Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub> (sodium thiosulfate) in their | ||
+ | BG11 | ||
+ | plates | ||
+ | and we could find literature to support this advice <a | ||
+ | href="https://jb.asm.org/content/171/10/5743.short" target="_blank">(Thiel | ||
+ | et al., 1989)</a>. This might seem like | ||
+ | a | ||
+ | small | ||
+ | addition, but it proved to be an essential factor, as after adding this | ||
+ | ingredient to our own | ||
+ | plates | ||
+ | we | ||
+ | were able to grow way better looking colonies, and not just that - the | ||
+ | colonies | ||
+ | started coming | ||
+ | up | ||
+ | after | ||
+ | just one day in our incubator. | ||
+ | |||
+ | The reason is simple: When autoclaving agar that contains phosphates, | ||
+ | reactive | ||
+ | oxygen species | ||
+ | such | ||
+ | as | ||
+ | H<sub>2</sub>O<sub>2</sub> can be formed, which can drastically hinder | ||
+ | bacterial | ||
+ | growth | ||
+ | <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249246/" | ||
+ | target="_blank">(Tanaka et al., 2014)</a>. The | ||
+ | thiosulfate from Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub> is oxidized as | ||
+ | H<sub>2</sub>O<sub>2</sub> is reduced to 2 H<sub>2</sub>O - so clearly | ||
+ | thiosulfate can | ||
+ | act as a reducing agent, eliminating reactive oxygen species from the | ||
+ | medium. | ||
+ | Presumably the | ||
+ | missing sodium thiosulfate in our BG11 agar was not the sole reason we did | ||
+ | not | ||
+ | get the desired | ||
+ | results on our plate, but in combination with the toxicity of Cas12a (though | ||
+ | less toxic than | ||
+ | Cas9, it still exhibits nuclease activity, cutting the DNA of the bacteria) | ||
+ | the | ||
+ | pressure was | ||
+ | too high for most of the cells. With our new found ingredient we were | ||
+ | certain to | ||
+ | get it right | ||
+ | this time and set the whole process in motion, this time with BG11 plates | ||
+ | including sodium | ||
+ | thiosulfate. In one huge experiment we tried a wide variety of different | ||
+ | conjugation | ||
+ | approaches, following the instructions of multiple papers ( <a | ||
+ | href=https://www.nature.com/articles/srep39681>Ungerer and Pakrasi, | ||
+ | 2016</a>; <a href=https://www.nature.com/articles/srep08132>Yu <i>et | ||
+ | al.</i>, 2015</a> ; <a | ||
+ | href=https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0514-7> | ||
+ | Wendt <i>et al.</i>, 2016</a>). We are currently screening for correct | ||
+ | edits | ||
+ | and are certain to hold | ||
+ | a naturally competent strain in our hands in a matter of days.</p> | ||
+ | <figure Style="text-align:center"> | ||
+ | <img style="height: 50ex; width: 60ex" | ||
+ | src=https://static.igem.org/mediawiki/2019/f/ff/T--Marburg--Conjugation_UTEX2973_pSL2680.png | ||
+ | alt="latest conjugation of UTEX2973 with the plasmid pSL2680."> | ||
+ | <figcaption> | ||
+ | Fig.4 : Latest conjugation of UTEX2973 with the plasmid pSL2680. | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <br> | ||
+ | <br> | ||
+ | |||
+ | </p> | ||
+ | <p> | ||
+ | <u>CRISPR gene editing</u> | ||
+ | </p> | ||
+ | <p> | ||
+ | Although CRISPR/Cas systems have been discussed as incredibly powerful tools in | ||
+ | genetic | ||
+ | engineering, | ||
+ | they have | ||
+ | not yet been widely used in cyanobacterial research, which is why we set out to | ||
+ | implement such a | ||
+ | system, based | ||
+ | on CRISPR/Cas12a, into our <a href="https://2019.igem.org/Team:Marburg/Part_Collection" | ||
+ | target="_blank">Green | ||
+ | Expansion</a> of the Marburg Collection. | ||
+ | <br> | ||
+ | As CRISPR/Cas12a has already been reported to work in <i> S.elongatus</i> UTEX 2973 | ||
+ | <a href=https://www.nature.com/articles/srep39681> (Ungerer and Pakrasi, 2016)</a>, we | ||
+ | were sure that it could be transformed into a Golden Gate Assembly compatible | ||
+ | version, allowing for more flexible <a | ||
+ | href="https://2019.igem.org/Team:Marburg/Design#strain_engineering" | ||
+ | target="_blank">design</a> considerations. | ||
+ | While we started the cloning processes needed to change the existing vector into the | ||
+ | phytobrick | ||
+ | standard, we | ||
+ | tried the vector at hand ourselves, in order to assess its usefulness. | ||
+ | Following the given protocols we constructed a CRISPR/Cas12a vector harboring a | ||
+ | crRNA | ||
+ | and repair | ||
+ | template | ||
+ | designed to revert the point mutation in the <i> pilN </i> gene of our <i> | ||
+ | S.elongatus</i> strain. | ||
+ | After a | ||
+ | few | ||
+ | initial problems we were able to get conjugants and are currently screening for | ||
+ | those | ||
+ | containing | ||
+ | the | ||
+ | desired | ||
+ | edit - more on this approach can be found in the natural competence section of our | ||
+ | results. | ||
+ | <br> | ||
+ | In order to modularize this system we built different parts for our genetic toolbox. | ||
+ | First of all | ||
+ | we | ||
+ | created | ||
+ | a | ||
+ | lvl0 part of the Cas12a protein by amplifying the sequence from the pSL2680 plasmid, | ||
+ | including | ||
+ | overhangs | ||
+ | that | ||
+ | enabled us to clone the PCR product into a lvl0 acceptor vector. | ||
+ | |||
+ | <figure Style="text-align:center"> | ||
+ | <img style="height: 45ex; width: 60ex" | ||
+ | src=https://static.igem.org/mediawiki/2019/a/ae/T--Marburg--Cas12a_CDS_lvl0.png | ||
+ | alt="map"> | ||
+ | <figcaption> | ||
+ | Fig. 5: The plasmid map for the coding sequence of the lvl0 construct. | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <p> | ||
+ | Sequencing results proved, that this crucial part was correctly assembled, ready | ||
+ | to | ||
+ | be used in lvl | ||
+ | 1 | ||
+ | constructs | ||
+ | - which we promptly did, using the following lvl0 parts: | ||
+ | pMC0_1_03 + pMC0_2_03 + pMC0_3_07 + pMC0_4_33 + pMC0_5_07 + pMC0_6_17. </p> | ||
+ | <figure Style="text-align:center"> | ||
+ | <img style="height: 45ex; width: 60ex" | ||
+ | src=https://static.igem.org/mediawiki/2019/a/ae/T--Marburg--Cas12a_CDS_lvl0.png | ||
+ | alt="map"> | ||
+ | <figcaption> | ||
+ | Fig. 6: Sequencing result of the coding sequence of the Cas12a protein. | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <p> | ||
+ | The chosen promoter is a rather weak one, so that overproduction of Cas12a is | ||
+ | prevented, leading | ||
+ | to | ||
+ | less | ||
+ | toxicity in the cells. </p> | ||
+ | <figure Style="text-align:center"> | ||
+ | <img style="height: 45ex; width: 65ex" | ||
+ | src=https://static.igem.org/mediawiki/2019/7/74/T--Marburg--Cas12a_CDS_lvl1.png | ||
+ | alt="map"> | ||
+ | <figcaption> | ||
+ | Fig. 7: The lvl1 construct of the Cas12a protein was built with a weaker | ||
+ | promoter to reduce | ||
+ | toxic | ||
+ | effects. | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <br> | ||
+ | <p> | ||
+ | Having built this construct, we continued to build the other missing part: the | ||
+ | crRNA. | ||
+ | The design of the pSL2680 plasmid was mostly kept the same, but in order to have | ||
+ | an | ||
+ | easy and cheap | ||
+ | selection | ||
+ | method we switched the lacZ cassette with a GFP cassette. </p> | ||
+ | <figure Style="text-align:center"> | ||
+ | <img style="height: 40ex; width: 90ex" | ||
+ | src=https://static.igem.org/mediawiki/2019/7/7e/T--Marburg--Cas12a_crRNA_lvl0.png | ||
+ | alt="crRNA lvl0"> | ||
+ | <figcaption> | ||
+ | Fig. 8: lvl0 design for the crRNA. The spacers can be exchanged with even | ||
+ | more | ||
+ | gRNAs to allow | ||
+ | for | ||
+ | multiplex genome editing. | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <br> | ||
+ | |||
+ | <p> | ||
+ | We could show the correct assembly of this part - everything was as we planned | ||
+ | in | ||
+ | our <a href="https://2019.igem.org/Team:Marburg/Design#strain_engineering" | ||
+ | target="_blank">design</a> | ||
+ | meaning that we had all the parts in our MoClo standard. </p> | ||
+ | <figure Style="text-align:center"> | ||
+ | <img style="height: 40ex; width: 80ex" | ||
+ | src=https://static.igem.org/mediawiki/2019/3/30/T--Marburg--Cas12a_crRNA_lvl0_seq.png | ||
+ | alt="[Fig XX seq results of crRNA lvl0],"> | ||
+ | <figcaption> | ||
+ | Fig. 9: Sequencing result of the lvl0 construct of the crRNA | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <br> | ||
+ | <p> | ||
+ | As the whole system is built for modular cloning in the PhytoBrick syntax, it is | ||
+ | possible to | ||
+ | freely | ||
+ | exchange | ||
+ | the | ||
+ | parts around the Cas12a and crRNA parts. This enables the use of different | ||
+ | promoters, allowing for | ||
+ | easy | ||
+ | screening: Constructs with weaker promoters in front of the Cas12a gene would | ||
+ | lead | ||
+ | to less gene | ||
+ | expression | ||
+ | and | ||
+ | therefore lower toxicity of the whole system. The free exchange of these | ||
+ | promoter | ||
+ | parts can | ||
+ | consequently be | ||
+ | used | ||
+ | for the creation of a library in order to look for the perfectly fitting | ||
+ | promoter | ||
+ | for this system. | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | Successfully creating these invaluable parts, we were able to establish a | ||
+ | workflow | ||
+ | for faster | ||
+ | cloning in <i> | ||
+ | S.elongatus</i>. | ||
+ | As our system is modularized, it is possible to easily exchange the GFP cassette | ||
+ | for | ||
+ | the desired | ||
+ | crRNA, | ||
+ | which | ||
+ | can be done in a single reaction, further simplifying the cloning process of | ||
+ | CRISPR/Cas12a | ||
+ | constructs. | ||
+ | As shown <a href=https://2019.igem.org/Team:Marburg/Design#strain_engineering> | ||
+ | before</a> , the cloning process with the pSL2680 can take over a week, is | ||
+ | tedious work and is accompanied by another couple of days waiting for colonies. | ||
+ | In comparison, our system enables for efficient cloning in only four days: On | ||
+ | the first day the construct is assembled in a Golden Gate reaction, which is | ||
+ | thereafter transformed into <i>E.coli</i>. The next day | ||
+ | colonies can be picked, | ||
+ | inoculated and the construct can be | ||
+ | extracted in the evening. On the third day it can be transformed into <i> | ||
+ | S.elongatus</i> - | ||
+ | and on the fourth day colonies can be screened. | ||
+ | The missing piece to apply an edit is the repair template. Skimming through | ||
+ | literature, we | ||
+ | noticed | ||
+ | that | ||
+ | transformation of linear DNA fragments into <i>S.elongatus</i> is supposedly | ||
+ | more efficient | ||
+ | than | ||
+ | the | ||
+ | transformation of whole plasmids <a | ||
+ | href=https://doi.org/10.1016/j.biori.2017.09.001> (Almeida <i>et | ||
+ | al.</i>, 2017)</a> - and we were able to verify this fact in our own | ||
+ | experiments. This further simplifies our above mentioned workflow, as we are | ||
+ | able | ||
+ | to simply PCR the needed repair template from a DNA sequence and use the PCR | ||
+ | product for | ||
+ | transformation into <i> | ||
+ | S.elongatus</i>. Our toolbox has a | ||
+ | special feature that can be used for exactly this workflow: a NotI cutting | ||
+ | site | ||
+ | can be found | ||
+ | in | ||
+ | our | ||
+ | constructs, | ||
+ | which is used to linearize them, so that they can be more efficiently | ||
+ | transformed. | ||
+ | <br> | ||
+ | We are more than certain that our modular CRISPR/Cas12a proves to be an | ||
+ | invaluable | ||
+ | contribution | ||
+ | to the | ||
+ | tools | ||
+ | available in cyanobacterial research, especially for the Golden Gate | ||
+ | community, | ||
+ | which is | ||
+ | growing | ||
+ | bigger | ||
+ | and | ||
+ | bigger every year - also thanks to the iGEM headquarters finally integrating | ||
+ | the | ||
+ | TypeIIS | ||
+ | standard into | ||
+ | the | ||
+ | competition! | ||
+ | <br> | ||
+ | <br> | ||
+ | </p> | ||
+ | <p> | ||
+ | <u>Cyanobacterial shuttle vectors </u> | ||
+ | </p> | ||
+ | <p>As we have already clarified in the <a | ||
+ | href="https://2019.igem.org/Team:Marburg/Description#strain_engineering" | ||
+ | target="_blank">description</a> part, self replicating shuttle vectors are | ||
+ | essential | ||
+ | for | ||
+ | many | ||
+ | workflows, as the gene expression levels are higher and non of the tedious | ||
+ | selection | ||
+ | processes that | ||
+ | come | ||
+ | with | ||
+ | genomic integrations have to be done. </p><br> | ||
+ | |||
+ | <p>On our road to the modular vector we were seeking, we firstly cured our own S. | ||
+ | elongatus UTEX 2973 | ||
+ | strain of its | ||
+ | pANS plasmid. This was done by transforming the pAM4787 vector, which holds a | ||
+ | spectinomycin | ||
+ | resistance | ||
+ | as well as a | ||
+ | YFP cassette | ||
+ | <a href=https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000377> | ||
+ | (Chen <i>et al.</i>, | ||
+ | 2016)</a>. Due to plasmid incompatibility - explained here in our <a | ||
+ | href="https://2019.igem.org/Team:Marburg/Design#strain_engineering" | ||
+ | target="_blank">design</a> section - and because antibiotic pressure is | ||
+ | applied, | ||
+ | the pANS plasmid was over time | ||
+ | cured | ||
+ | from the | ||
+ | strain, which then just kept the pAM4787 plasmid. Transformation was done by | ||
+ | conjugation with the | ||
+ | pRK2013 plasmid in | ||
+ | DH5ɑ and the pAM4787 in HB101. Both were grown to an OD600≈0.5, washed in LB and | ||
+ | mixed with <i>S. | ||
+ | elongatus</i> which was | ||
+ | grown to late exponential phase and then washed in BG11. | ||
+ | We could clearly show, that the conjugant strain bears the pAM4787 plasmid if | ||
+ | selective pressure | ||
+ | is | ||
+ | held up.</p> | ||
+ | |||
+ | <figure Style="text-align:center"> | ||
+ | <img style="height: 65ex; width: 50ex" | ||
+ | src=https://static.igem.org/mediawiki/2019/e/eb/T--Marburg--YFPconstructConjugantFACS.png | ||
+ | alt=" https://static.igem.org/mediawiki/2019/e/eb/T--Marburg--YFPconstructConjugantFACS.png"> | ||
+ | <figcaption> | ||
+ | Fig. 10: Cell counts of conjugant strain. The y axis shows relative YFP | ||
+ | fluorescence and the x | ||
+ | axis relative autofluorescence. | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <p>This was followed by us starting to culture the pAM4787 bearing strain without | ||
+ | antibiotics again, slowly removing selective pressure from the cells. As the | ||
plasmid | plasmid | ||
− | + | does not give | |
− | + | them any other | |
− | + | advantage and is probably just more metabolic burden due to the constantly | |
− | + | produced | |
− | + | YFP proteins it | |
+ | is | ||
+ | slowly being | ||
+ | lost. | ||
+ | We could prove this in multiple setups: with the flow cytometry device we were | ||
+ | kindly granted access | ||
+ | to we could | ||
+ | clearly show the missing YFP signal in the cured <i>S. elongatus </i>strain and | ||
+ | logically this could also be observed over our UV table.</p> | ||
+ | |||
+ | <figure Style="text-align:center"> | ||
+ | <img style="height: 65ex; width: 50ex" | ||
+ | src=https://static.igem.org/mediawiki/2019/c/c4/T--Marburg--CuredStrainsFACS.png | ||
+ | alt="https://static.igem.org/mediawiki/2019/c/c4/T--Marburg--CuredStrainsFACS.png "> | ||
+ | <img style="height: 65ex; width: 50ex" | ||
+ | src=https://static.igem.org/mediawiki/2019/d/da/T--Marburg--CuredStrainUV.png | ||
+ | alt="https://static.igem.org/mediawiki/2019/d/da/T--Marburg--CuredStrainUV.png"> | ||
+ | <figcaption> Fig. 11 | ||
+ | a) Cell counts of cured strain. The y axis shows relative YFP fluorescence | ||
+ | and | ||
+ | the x axis | ||
+ | relative | ||
+ | autofluorescence | ||
+ | b) Comparison of the fluorescence signal of the transformed (left) and cured | ||
+ | (right) strain. | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Furthermore we performed colony PCRs as a test. We sent our plasmid-free strain | ||
+ | to | ||
+ | Next Generation | ||
+ | Sequencing in order to ensure that the strain really has lost the | ||
+ | pANS plasmid.</p> | ||
+ | <figure Style="text-align:center"> | ||
+ | <img style="height: 65ex; width: 50ex" | ||
+ | src=https://static.igem.org/mediawiki/2019/e/e9/T--Marburg--ColonyPCRcuredStrain.jpg | ||
+ | alt="gel pcr"> | ||
+ | <figcaption> Fig. 12: Colony PCR of the wild type, the conjugated and the cured | ||
+ | strain. | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | |||
+ | <p>Our next step was the characterization of the cyanobacterial shuttle vector | ||
+ | mentioned | ||
+ | in our <a href="https://2019.igem.org/Team:Marburg/Design#strain_engineering" | ||
+ | target="_blank"> design </a> section. In an extensive flow cytometry | ||
+ | experiment we assessed the fluorescence of a transformed YFP-construct in our | ||
+ | cured | ||
+ | strain, showing | ||
+ | that the shuttle vector with the minimal replication element can be maintained | ||
in | in | ||
− | + | <i>S. elongatus | |
− | + | </i> UTEX | |
− | + | 2973. </p><br> | |
− | + | ||
− | + | <p>After another four weeks of cultivation we looked at our | |
+ | cultures again on the UV table to check if fluorescence was still present and | ||
the | the | ||
− | + | high intensity | |
− | + | of | |
− | + | fluorescence | |
− | + | proved to us, that the plasmid is still stably replicated in our strain, showing | |
− | + | us, | |
− | + | that the | |
− | + | minimal replication | |
− | + | element does indeed work in our strain. | |
− | we | + | For further analysis we performed qPCR with this transformed strain, in order to |
− | were | + | check the copy |
− | + | number of the | |
− | + | vector. We used the copy number of pANL as a reference, which is supposedly at | |
− | + | ~2,6 | |
− | + | copies per | |
+ | chromosome | ||
+ | <a href=https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000377> | ||
+ | (Chen <i>et al.</i>, | ||
+ | 2016)</a>. Our data shows a ~4,5 times higher copy number relative to pANL, | ||
+ | meaning that the | ||
+ | construct is maintained with approximately 11,7 copies per chromosome.</p> | ||
+ | |||
+ | <p> | ||
+ | For further analysis of this part (<a style="padding: 0" | ||
+ | href=" http://parts.igem.org/Part:BBa_K3228069" | ||
+ | target="_blank">BBa_K3228069</a>) we performed a | ||
+ | Quantitative-Polymerase-Chain-Reaction (qPCR) with this transformed strain, in | ||
+ | order | ||
+ | to check the copy number of the vector. This proved to be difficult in | ||
+ | Cyanobacteria, due to variation in genome copy number (<a style="padding: 0" | ||
+ | href=" https://onlinelibrary.wiley.com/doi/full/10.1111/j.1574-6968.2011.02368.x?sid=nlm%3Apubmed" | ||
+ | target="_blank">Griese <i>et al.</i>, 2011</a>; <a style="padding: 0" | ||
+ | href=" https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2427279/" target="_blank"> | ||
+ | Chen <i>et al.</i>, 2012</a>). | ||
+ | To overcome this problem, we noticed that the copynumber of pANL stays rather | ||
+ | stable | ||
+ | with 2,6 copies per chromosome (<a style="padding: 0" | ||
+ | href=" https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000377" | ||
+ | target="_blank">Chen <i>et al.</i>, 2016</a>). | ||
+ | Therefore we performed the qPCR with pANL as an additional target. For each | ||
+ | target, | ||
+ | we chose three different primer pairs, with known efficiency. Five technical and | ||
+ | two | ||
+ | biological replicates were used. The samples were normalised to the chromosome | ||
+ | and | ||
+ | pANL was used to identify the total copy number per chromosome (figure 14). Our | ||
+ | data | ||
+ | shows a ~4,5 times higher copy number relative to pANL, meaning that the | ||
+ | construct | ||
+ | is maintained with approximately 11,7 copies per chromosome. This is comparable | ||
+ | with | ||
+ | the copynumber of pANS in <a style="padding: 0" | ||
+ | href=" https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000377" | ||
+ | target="_blank"> Chen <i>et al.</i> 2016</a>. | ||
+ | </p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <figure Style="text-align:center"> | ||
+ | <img style="height: 65ex; width: 100ex" | ||
+ | src="https://static.igem.org/mediawiki/2019/d/d6/T--Marburg--Parts--qPCR-Lvl1.png" | ||
+ | alt="Copynumber Evaluation of Ori-part via qPCR"> | ||
+ | <figcaption> Fig. 14: Copynumber Evaluation of Ori-part via qPCR | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <p>Additionally we measured the fluorescence signals in a plate reader at different | ||
+ | optical | ||
+ | densities | ||
+ | and could again | ||
+ | confirm high fluorescence signals, indicating strong gene expression in | ||
+ | constructs | ||
+ | built | ||
+ | around | ||
+ | this replication | ||
+ | element. </p> | ||
+ | <figure Style="text-align:center"> | ||
+ | <img style="height: 55ex; width: 60ex" | ||
+ | src=https://static.igem.org/mediawiki/2019/f/f0/T--Marburg--results_yfp_pam_4787_6_replicates.jpg | ||
+ | alt="diagramm"> | ||
+ | <figcaption> Fig. 12:YFP fluorescence at different optical densities. | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | |||
+ | |||
+ | <p>All this data confirms that the construct actually works and can be reliably used | ||
as | as | ||
− | + | a | |
− | + | cyanobacterial shuttle | |
− | + | vector, proving that BBa_K3228069 works as intended, thus functioning as our <a | |
− | + | href="parts.igem.org/Part:BBa_K3228069" target="_blank"> validated part</a>. | |
− | + | This assumption is solidified by all our sequence data, showing that the shuttle | |
− | + | vectors were | |
− | + | completely assembled | |
− | + | as planned in our design section. </p> | |
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− | + | <div class="sub" onclick="popup('marburg_collection')"> | |
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− | + | <div class="sub-header"> | |
− | + | <h1> | |
− | + | M A R B U R G <br> C O L L E C T I O N  2.0 | |
− | + | </h1> | |
− | + | <hr> | |
− | + | </div> | |
+ | <div class="sub-content"> | ||
+ | <div> | ||
+ | We expanded the Marburg Collection by adding the Green expansion and the first MoClo | ||
+ | compatible | ||
+ | shuttle | ||
+ | vector for Cyanobacteria. | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
− | <div | + | <div id="marburg_collection" class="popup"> |
− | + | <div class="popup-container"> | |
− | + | <div class="popup-header"> | |
− | + | <h1 class="title">Marburg Collection 2.0</h1> | |
− | + | <button type="button" onclick="hide('marburg_collection')">X</button> <!-- nummerieren--> | |
− | + | </div> | |
− | + | <div class="popup-content" style="text-align: justify;"> | |
− | + | <section class="section"> | |
− | + | <p><u>Overview over the expansion of the Marburg Collection:</u></p> | |
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− | + | <p>We added 55 new parts to the Marburg Collection, adding several new features such as | |
− | + | the | |
− | + | Green | |
− | + | expansion, | |
− | + | including a | |
− | + | kit for the Modularized Engineering of Genome Areas (M.E.G.A.) and the first MoClo | |
− | + | compatible shuttle | |
− | + | vector for cyanobacteria. | |
− | + | Additionally, we offer a set of reporters suitable for characterization of BioBricks | |
− | + | in | |
− | + | cyanobacteria | |
− | + | and | |
− | + | ribozymes for a more | |
− | + | stable and species independent transcription. We also provide standardized | |
− | + | measurement | |
− | + | vectors that | |
− | + | were | |
− | + | generated using our | |
− | + | designed placeholders.</p><br> | |
− | + | ||
− | + | ||
− | + | <figure style="text-align:center"> | |
+ | <img style="height: 400px; width: 1000px;" | ||
+ | src="https://static.igem.org/mediawiki/2019/2/2b/T--Marburg--Toolbox_table.svg" | ||
+ | alt="Overview over the expansion of the Marburg Collection"> | ||
+ | <figcaption style="max-width: 2400px; text-align: center"> | ||
+ | Fig.1: Overview over the expansion of the Marburg Collection | ||
+ | </figcaption> | ||
+ | </figure><br> <br> | ||
+ | |||
+ | |||
+ | <p><u>Overview over the different expansions in the Marburg Collection 2.0</u></p> | ||
+ | <p>To give a better overview we show here the different expansions we added to the | ||
+ | Marburg | ||
+ | Collection:<br> | ||
+ | |||
+ | <section style="margin-top: 11vh; padding-bottom: 1.5em;"> | ||
+ | <h1 class="title"> | ||
+ | Parts | ||
+ | </h1> | ||
+ | <table class="table is-fullwidth; parts-table"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/5/5b/T--Marburg--5%27Homology.svg" | ||
+ | alt="5'Homology"> | ||
+ | </th> | ||
+ | <th> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/3/3a/T--Marburg--Terminator.svg" | ||
+ | alt="Termiantor"> | ||
+ | </th> | ||
+ | <th> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/1/1e/T--Marburg--Antibiotic_Resistance.svg" | ||
+ | alt="AntibioticResistance"> | ||
+ | </th> | ||
+ | <th> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/3/30/T--Marburg--3%27Homology.svg" | ||
+ | alt="3'Homology"> | ||
+ | </th> | ||
+ | <th> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/f/fc/T--Marburg--Shuttle_Vector.svg" | ||
+ | alt="ShuttleVector"> | ||
+ | <th> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/5/51/T--Marburg--CDS.svg" | ||
+ | alt="CDS"> | ||
+ | </th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <div style="display: flex; flex-direction: row; margin-top: 25px"> | ||
+ | <div> | ||
+ | <div class="content"> | ||
+ | <ul> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228000">K3228000</a> | ||
+ | (aNSo1 | ||
+ | integration up) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228001">K3228001</a> | ||
+ | (aNSo2 | ||
+ | integration up) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228020">K3228020</a> | ||
+ | (NS1 up) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228021">K3228021</a> | ||
+ | (NS2 up) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228022">K3228022</a> | ||
+ | (NS3 up) | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </td> | ||
+ | <td> | ||
+ | <div style="display: flex; flex-direction: row; margin-top: 25px"> | ||
+ | <div> | ||
+ | <div class="content"> | ||
+ | <ul> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228004">K3228004</a> | ||
+ | (shortTB0010) | ||
+ | </li> | ||
+ | <li> <a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228005">K3228005</a> | ||
+ | (shortTB0015) | ||
+ | </li> | ||
+ | <li> <a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228006">K3228006</a> | ||
+ | (shortTB1002) | ||
+ | </li> | ||
+ | <li> <a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228007">K3228007</a> | ||
+ | (shortTB1003) | ||
+ | </li> | ||
+ | <li> <a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228008">K3228008</a> | ||
+ | (shortTB1004) | ||
+ | </li> | ||
+ | <li> <a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228009">K3228009</a> | ||
+ | (shortTB1005) | ||
+ | </li> | ||
+ | <li> <a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228010">K3228010</a> | ||
+ | (shortTB1006) | ||
+ | </li> | ||
+ | <li> <a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228011">K3228011</a> | ||
+ | (shortTB1007) | ||
+ | </li> | ||
+ | <li> <a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228012">K3228012</a> | ||
+ | (shortTB1008) | ||
+ | </li> | ||
+ | <li> <a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228013">K3228013</a> | ||
+ | (shortTB1009) | ||
+ | </li> | ||
+ | <li> <a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228014">K3228014</a> | ||
+ | (shortTB1010) | ||
+ | </li> | ||
+ | <li> <a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228015">K3228015</a> | ||
+ | (shortDummy) | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </td> | ||
+ | <td> | ||
+ | <div style="display: flex; flex-direction: row; margin-top: 25px"> | ||
+ | <div> | ||
+ | <div class="content"> | ||
+ | <ul> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228016">K3228016</a> | ||
+ | (SpecRes_short) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228017">K3228017</a> | ||
+ | (CmlRes_short) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228018">K3228018</a> | ||
+ | (TetRes_short) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228019">K3228019</a> | ||
+ | (KanRes_short) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228027">K3228027</a> | ||
+ | (GenRes_short) | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </td> | ||
+ | <td> | ||
+ | <div style="display: flex; flex-direction: row; margin-top: 25px"> | ||
+ | <div> | ||
+ | <div class="content"> | ||
+ | <ul> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228002">K3228002</a> | ||
+ | (aNSo1 integration down) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228003">K3228003</a> | ||
+ | (aNSo2 integration down) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228023">K3228023</a> | ||
+ | (NS1 down) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228024">K3228024</a> | ||
+ | (NS2 down) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228025">K3228025</a> | ||
+ | (NS3 down) | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </td> | ||
+ | <td> | ||
+ | <div style="display: flex; flex-direction: row; margin-top: 25px"> | ||
+ | <div> | ||
+ | <div class="content"> | ||
+ | <ul> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228026">K3228026</a> | ||
+ | (oriT integration) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228069">K3228069</a> | ||
+ | (panS SpecRes LVL1) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228089">K3228089</a> | ||
+ | (panS KanRes LVL2) | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </td> | ||
+ | <td> | ||
+ | <div style="display: flex; flex-direction: row; margin-top: 25px"> | ||
+ | <div> | ||
+ | <div class="content"> | ||
+ | <ul> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228051">K3228051</a> | ||
+ | (Limonene | ||
+ | Synthase) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228052">K3228052</a> | ||
+ | (Farnesen | ||
+ | Synthase) | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <table class="table is-fullwidth; parts-table"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th style="text-align: center;"> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/2/28/T--Marburg--Ribo_Insulator.svg" | ||
+ | alt="RiboInsulator"> | ||
+ | </th> | ||
+ | <th> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/1/15/T--Marburg--Spaceholder.svg" | ||
+ | alt="Spaceholder"> | ||
+ | </th> | ||
+ | <th> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/4/4e/T--Marburg--Measurement_Vector.svg" | ||
+ | alt="Measurement"> | ||
+ | </th> | ||
+ | <th> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/3/3c/T--Marburg--CRISPR.svg" | ||
+ | alt="CRISPR"> | ||
+ | </th> | ||
+ | <th> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/4/4e/T--Marburg--Luminescence_Reporter.svg" | ||
+ | alt="LuminescenceReporter"> | ||
+ | </th> | ||
+ | <th> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/1/15/T--Marburg--Fluorescence_Reporter.svg" | ||
+ | alt="FluorescenceReporter"> | ||
+ | </th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <div style="display: flex; flex-direction: row; margin-top: 25px"> | ||
+ | <div> | ||
+ | <div class="content"> | ||
+ | <ul> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228054">K3228054</a> | ||
+ | (riboJ B0034) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228055">K3228055</a> | ||
+ | (riboJ B0034 noScar) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228056">K3228056</a> | ||
+ | (SarJ B0034) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228057">K3228057</a> | ||
+ | (PlmJ B0034) | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </td> | ||
+ | <td> | ||
+ | <div style="display: flex; flex-direction: row; margin-top: 25px"> | ||
+ | <div> | ||
+ | <div class="content"> | ||
+ | <ul> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228060">K3228060</a> | ||
+ | (PRO placeholder) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228061">K3228061</a> | ||
+ | (RBS placeholder) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228062">K3228062</a> | ||
+ | (CDS placeholder) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228063">K3228063</a> | ||
+ | (TER placeholder) | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </td> | ||
+ | <td> | ||
+ | <div style="display: flex; flex-direction: row; margin-top: 25px"> | ||
+ | <div> | ||
+ | <div class="content"> | ||
+ | <ul> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228073">K3228073</a> | ||
+ | (Promotor entry vector) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228074">K3228074</a> | ||
+ | (BS entry vector) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228075">K3228075</a> | ||
+ | (CDS entry vector) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228090">K3228090</a> | ||
+ | (Terminator entry vector) | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </td> | ||
+ | <td> | ||
+ | <div style="display: flex; flex-direction: row; margin-top: 25px"> | ||
+ | <div> | ||
+ | <div class="content"> | ||
+ | <ul> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228100">K3228100</a> | ||
+ | (cpf1) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228101">K3228101</a> | ||
+ | (crRNA-GFP dropout) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228103">K3228103</a> | ||
+ | (cpf1+crRNA-GFP dropout) | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </td> | ||
+ | <td> | ||
+ | <div style="display: flex; flex-direction: row; margin-top: 25px"> | ||
+ | <div> | ||
+ | <div class="content"> | ||
+ | <ul> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228040">K3228040</a> | ||
+ | (NanoLuc) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228041">K3228041</a> | ||
+ | (NanoLuc S.e.) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228042">K3228042</a> | ||
+ | (TeLuc (S.e.)) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228043">K3228043</a> | ||
+ | (Antares S.e.) | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </td> | ||
+ | <td> | ||
+ | <div style="display: flex; flex-direction: row; margin-top: 25px"> | ||
+ | <div> | ||
+ | <div class="content"> | ||
+ | <ul> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228044">K3228044</a> | ||
+ | (eYFP S.e.) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228045">K3228045</a> | ||
+ | (sYFP2 S.e.) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228046">K3228046</a> | ||
+ | (phluorin2 S.e.) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228047">K3228047</a> | ||
+ | (rxYFP S.e.) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228048">K3228048</a> | ||
+ | (sfGFP S.e.) | ||
+ | </li> | ||
+ | <li><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3228049">K3228049</a> | ||
+ | (mTurquoise S.e.) | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | </section> | ||
+ | |||
+ | </p><br><br> | ||
+ | <p><u>Sequencing results of the lvl0 parts</u></p> | ||
+ | <p>We built and validated 55 new BioBricks this year. They are all listed in the | ||
+ | Registry of | ||
+ | Standard | ||
+ | Biological Parts | ||
+ | (Part range BBa_3228000 to BBa_32280103). All lvl0 Parts were validated by complete | ||
+ | sequencing.<br> | ||
+ | |||
+ | <figure style="text-align:center"> | ||
+ | <img style="height: 200px; width: 600px;" | ||
+ | src="https://static.igem.org/mediawiki/2019/d/d4/T--Marburg--anso-lvl_0_front.jpg" | ||
+ | alt="aNSo-lvl-0-front"> | ||
+ | <figcaption style="max-width: 2400px; text-align: center"> | ||
+ | Fig.7: aNSo-lvl-0-front | ||
+ | </figcaption> | ||
+ | </figure><br> | ||
+ | |||
+ | <figure style="text-align:center"> | ||
+ | <img style="height: 200px; width: 600px;" | ||
+ | src="https://static.igem.org/mediawiki/2019/a/ab/T--Marburg--anso_lvl_0_end.jpg" | ||
+ | alt="aNSo-lvl-0-end"> | ||
+ | <figcaption style="max-width: 2400px; text-align: center"> | ||
+ | Fig.8: aNSo-lvl-0-end | ||
+ | </figcaption> | ||
+ | </figure><br> | ||
+ | |||
+ | </p><br><br> | ||
+ | <p><u>Building constructs to test the lethality of origin of transfer</u></p> | ||
+ | <p>If plasmids reach a certain size normal transformation protocols are not feasible | ||
+ | anymore | ||
+ | to bring | ||
+ | the | ||
+ | plasmid into | ||
+ | the host.<br> | ||
+ | For the transformation of such huge megaplasmids we designed an “origin of transfer” | ||
+ | BioBrick that | ||
+ | makes | ||
+ | it possible | ||
+ | to directly transport plasmids of any size from one species to another. To test if | ||
+ | this | ||
+ | sequence would | ||
+ | result in any | ||
+ | toxicity in a genomic context (source things where genome parts can be exchanged by | ||
+ | integrating such | ||
+ | sequences) we built | ||
+ | it into an integration vector. | ||
+ | </p><br><br> | ||
+ | <p><u>Sequencing results of the lvl1 parts for modularized genome integrations</u></p> | ||
+ | <p>We successfully build two integration cassettes from our rationally designed | ||
+ | artificial | ||
+ | neutral | ||
+ | integration sites | ||
+ | (a.N.S.o. 1 and 2) and verified them by sequencing. These parts contained the | ||
+ | “origin of | ||
+ | transfer” to | ||
+ | test | ||
+ | their lethality | ||
+ | in the aforementioned experiment.<br> | ||
+ | |||
+ | <figure style="text-align:center"> | ||
+ | <img style="height: 200px; width: 600px;" | ||
+ | src="https://static.igem.org/mediawiki/2019/2/2e/T--Marburg--anso_1_mit_Spec_LVL_1.jpg" | ||
+ | alt="aNSo1-lvl-1 with spec"> | ||
+ | <figcaption style="max-width: 2400px; text-align: center"> | ||
+ | Fig.9: aNSo1-lvl-1 with spec | ||
+ | </figcaption> | ||
+ | </figure><br> | ||
+ | |||
+ | <figure style="text-align:center"> | ||
+ | <img style="height: 200px; width: 600px;" | ||
+ | src="https://static.igem.org/mediawiki/2019/d/d6/T--Marburg--anso_2_mit_cml_LVL_1.jpg" | ||
+ | alt="anso2-lvl-1 with cml"> | ||
+ | <figcaption style="max-width: 2400px; text-align: center"> | ||
+ | Fig.10: anso2-lvl-1 with cml | ||
+ | </figcaption> | ||
+ | </figure><br> | ||
+ | </p><br> <br> | ||
+ | <p><u>Workflow to integrate a modularized integration cassette</u></p> | ||
+ | <p>We established a workflow on how to integrate a cassette - from lvl0 Parts to a | ||
+ | finished | ||
+ | change in | ||
+ | genome. With | ||
+ | UTEX 2973 this is possible in less than five days, while in PCC7942 the same | ||
+ | integration | ||
+ | would take a | ||
+ | whole | ||
+ | month.</p><br> | ||
+ | |||
+ | |||
+ | <figure style="text-align:center"> | ||
+ | <img style="height: 600px; width: 1800px;" | ||
+ | src="https://static.igem.org/mediawiki/2019/4/41/T--Marburg--Toolbox_Model_ANSOscreening.svg" | ||
+ | alt="anso wirkflow"> | ||
+ | <figcaption style="max-width: 2400px; text-align: center"> | ||
+ | Fig.11: Workflow for finding new neutral integration sites. | ||
+ | </figcaption> | ||
+ | </figure><br><br> | ||
+ | |||
+ | |||
+ | <p><u>Using the placeholder to build standard measurement vectors</u></p> | ||
+ | <p>We successfully used our placeholders to build and validate the standardized | ||
+ | measurement | ||
+ | vectors for | ||
+ | promoters, | ||
+ | ribosomal binding sites and coding sequences. We evaluated the cost and time savings | ||
+ | from a library | ||
+ | assembly with a | ||
+ | sample size of 25.<br> | ||
+ | Through our design decision to build placeholders we managed to cut the workload for | ||
+ | a | ||
+ | high throughput | ||
+ | assembly by around 72% | ||
+ | and the invested financial resources by 40 % with just a sample size of 25 | ||
+ | assemblies. | ||
+ | </p><br> | ||
+ | |||
+ | <div class="wrap-collabsible"> | ||
+ | <input id="collapsibleolol" class="toggle" type="checkbox"> | ||
+ | <label for="collapsibleolol" class="lbl-toggle"> Workload and cost for | ||
+ | placeholder</label> | ||
+ | <div class="collapsible-content"> | ||
+ | <div class="content-inner"> | ||
+ | <p> | ||
+ | <div> | ||
+ | <section style="margin-top: 11vh; padding-bottom: 1.5em;"> | ||
+ | <h1 class="title"> | ||
+ | Workload for placeholder | ||
+ | </h1> | ||
+ | <table class="table is-fullwidth"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th>Task</th> | ||
+ | <th>Time per sample [min]</th> | ||
+ | <th>Samples</th> | ||
+ | <th>Total</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <th>Assembly of entry vector (7 part assembly)</th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | 1 | ||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Golden Gate reaction</th> | ||
+ | <td> | ||
+ | 20 | ||
+ | </td> | ||
+ | <td> | ||
+ | 1 | ||
+ | </td> | ||
+ | <td> | ||
+ | 20 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Transformation</th> | ||
+ | <td> | ||
+ | 15 | ||
+ | </td> | ||
+ | <td> | ||
+ | 1 | ||
+ | </td> | ||
+ | <td> | ||
+ | 15 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Preparing 10 overnight cultures + purification | ||
+ | </th> | ||
+ | <td> | ||
+ | 15 | ||
+ | </td> | ||
+ | <td> | ||
+ | 10 | ||
+ | </td> | ||
+ | <td> | ||
+ | 150 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Test digesting</th> | ||
+ | <td> | ||
+ | 5 | ||
+ | </td> | ||
+ | <td> | ||
+ | 5 | ||
+ | </td> | ||
+ | <td> | ||
+ | 25 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Inserting Promoters (2 part assembly) (25 | ||
+ | BioBricks) | ||
+ | </th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | 25 | ||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Golden Gate reactions (simplified with a | ||
+ | mastermix) | ||
+ | </th> | ||
+ | <td> | ||
+ | 5 | ||
+ | </td> | ||
+ | <td> | ||
+ | 25 | ||
+ | </td> | ||
+ | <td> | ||
+ | 125 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Transformation</th> | ||
+ | <td> | ||
+ | 10 | ||
+ | </td> | ||
+ | <td> | ||
+ | 25 | ||
+ | </td> | ||
+ | <td> | ||
+ | 250 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Preparing overnight cultures + purification</th> | ||
+ | <td> | ||
+ | 15 | ||
+ | </td> | ||
+ | <td> | ||
+ | 50 | ||
+ | </td> | ||
+ | <td> | ||
+ | 750 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Total</th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | <b>1335</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>In hours</th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | <b>22,25</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><b>Workflow without placeholders</b></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>lvl1 Golden Gate Assembly</th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | 25 | ||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Golden Gate reaction</th> | ||
+ | <td> | ||
+ | 5 | ||
+ | </td> | ||
+ | <td> | ||
+ | 25 | ||
+ | </td> | ||
+ | <td> | ||
+ | 125 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Transformation</th> | ||
+ | <td> | ||
+ | 10 | ||
+ | </td> | ||
+ | <td> | ||
+ | 25 | ||
+ | </td> | ||
+ | <td> | ||
+ | 250 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Preparing 10 overnight cultures + purification | ||
+ | </th> | ||
+ | <td> | ||
+ | 15 | ||
+ | </td> | ||
+ | <td> | ||
+ | 250 | ||
+ | </td> | ||
+ | <td> | ||
+ | 3750 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Test digesting</th> | ||
+ | <td> | ||
+ | 5 | ||
+ | </td> | ||
+ | <td> | ||
+ | 125 | ||
+ | </td> | ||
+ | <td> | ||
+ | 625 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Total</th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | <b>4750</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>In hours</th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | <b>79,16666667</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Differences in hours</th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | <b>56,91666667</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Percentage saved</th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | <b>0,72</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | </section> | ||
+ | <section style="margin-top: 11vh; padding-bottom: 1.5em;"> | ||
+ | <h1 class="title"> | ||
+ | Cost for placeholder | ||
+ | </h1> | ||
+ | <table class="table is-fullwidth"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th>Major cost point</th> | ||
+ | <th>Price [€]</th> | ||
+ | <th>Samples</th> | ||
+ | <th>Total</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <th>With placeholder</th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><b>lvl1 Golden Gate mix for measurement vector | ||
+ | assembly</b></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | 1 | ||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>BsaI</th> | ||
+ | <td> | ||
+ | 2 | ||
+ | </td> | ||
+ | <td> | ||
+ | 1 | ||
+ | </td> | ||
+ | <td> | ||
+ | 2 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>BsmbI</th> | ||
+ | <td> | ||
+ | 2 | ||
+ | </td> | ||
+ | <td> | ||
+ | 1 | ||
+ | </td> | ||
+ | <td> | ||
+ | 2 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><b>Plasmid purification</b></th> | ||
+ | <td> | ||
+ | 2 | ||
+ | </td> | ||
+ | <td> | ||
+ | 5 | ||
+ | </td> | ||
+ | <td> | ||
+ | 10 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><b>Test digesting</b></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | 5 | ||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Enzyme</th> | ||
+ | <td> | ||
+ | 1 | ||
+ | </td> | ||
+ | <td> | ||
+ | 5 | ||
+ | </td> | ||
+ | <td> | ||
+ | 5 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Aggarose gel and ladder (approximation)</th> | ||
+ | <td> | ||
+ | 0,5 | ||
+ | </td> | ||
+ | <td> | ||
+ | 2,5 | ||
+ | </td> | ||
+ | <td> | ||
+ | 1,25 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Sequencing (estimated)</th> | ||
+ | <td> | ||
+ | 3 | ||
+ | </td> | ||
+ | <td> | ||
+ | 3 | ||
+ | </td> | ||
+ | <td> | ||
+ | 9 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><b>lvl1 Golden Gate mix for inserting the | ||
+ | promoter</b>></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | 25 | ||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>BsaI</th> | ||
+ | <td> | ||
+ | 2 | ||
+ | </td> | ||
+ | <td> | ||
+ | 25 | ||
+ | </td> | ||
+ | <td> | ||
+ | 50 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>BsmbI</th> | ||
+ | <td> | ||
+ | 2 | ||
+ | </td> | ||
+ | <td> | ||
+ | 25 | ||
+ | </td> | ||
+ | <td> | ||
+ | 50 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><b>Plasmid purification</b></th> | ||
+ | <td> | ||
+ | 2 | ||
+ | </td> | ||
+ | <td> | ||
+ | 50 | ||
+ | </td> | ||
+ | <td> | ||
+ | 100 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Sequencing</th> | ||
+ | <td> | ||
+ | 3 | ||
+ | </td> | ||
+ | <td> | ||
+ | 50 | ||
+ | </td> | ||
+ | <td> | ||
+ | 150 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><b>Total</b></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | <b>378,25</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><b>Without placeholder</b></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><b>lvl1 Golden Gate mix for measurement vector | ||
+ | assembly</b></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | 25 | ||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>BsaI</th> | ||
+ | <td> | ||
+ | 2 | ||
+ | </td> | ||
+ | <td> | ||
+ | 25 | ||
+ | </td> | ||
+ | <td> | ||
+ | 50 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>BsmbI</th> | ||
+ | <td> | ||
+ | 2 | ||
+ | </td> | ||
+ | <td> | ||
+ | 25 | ||
+ | </td> | ||
+ | <td> | ||
+ | 50 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><b>Plasmid purification</b></th> | ||
+ | <td> | ||
+ | 2 | ||
+ | </td> | ||
+ | <td> | ||
+ | 125 | ||
+ | </td> | ||
+ | <td> | ||
+ | 250 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><b>Test digesting</b></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | 125 | ||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Enzyme</th> | ||
+ | <td> | ||
+ | 1 | ||
+ | </td> | ||
+ | <td> | ||
+ | 125 | ||
+ | </td> | ||
+ | <td> | ||
+ | 125 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Aggarose gel and ladder (approximation)</th> | ||
+ | <td> | ||
+ | 0,5 | ||
+ | </td> | ||
+ | <td> | ||
+ | 62,5 | ||
+ | </td> | ||
+ | <td> | ||
+ | 31,25 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Sequencing (estimated)</th> | ||
+ | <td> | ||
+ | 3 | ||
+ | </td> | ||
+ | <td> | ||
+ | 41,625 | ||
+ | </td> | ||
+ | <td> | ||
+ | 124,875 | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><b>Total</b></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | <b>378,25</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><b>Difference</b></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | <b>251,875</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><b>Percentage saved</b></th> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
− | + | </td> | |
− | + | <td> | |
− | + | <b>0,4</b> | |
− | + | </td> | |
− | + | </tr> | |
− | + | </tbody> | |
− | + | </table> | |
− | + | </section> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | </ | + | |
</div> | </div> | ||
− | </ | + | </p> |
</div> | </div> | ||
− | + | </div> | |
− | + | </div> | |
− | + | <br><br> | |
− | + | <p><u>Construction of a promoter library with standard measuring vectors</u></p> | |
− | + | <p>We built a promoter library using our standard promoter measurement vector and 25 | |
− | + | BioBricks. | |
− | + | Here we show a list of all the BioBricks we used.<br> | |
− | + | <figure style="text-align:center"> | |
− | + | <img style="height: 400px; width: 600px;" | |
− | + | src="https://static.igem.org/mediawiki/2019/3/31/T--Marburg--promoter_library_1.jpg" | |
− | + | alt="Promoter library 1r"> | |
− | + | <figcaption style="max-width: 2400px; text-align: center"> | |
− | + | Fig.14: Promoter library 1 | |
− | + | </figcaption> | |
− | + | </figure><br> | |
− | + | ||
− | + | <figure style="text-align:center"> | |
− | + | <img style="height: 400px; width: 600px;" | |
− | + | src="https://static.igem.org/mediawiki/2019/1/1a/T--Marburg--promoter_library_2.jpg" | |
− | + | alt="Promoter library 2"> | |
− | + | <figcaption style="max-width: 2400px; text-align: center"> | |
− | + | Fig.15: Promoter library 2 | |
− | + | </figcaption> | |
− | + | </figure><br> | |
− | + | ||
− | + | </p><br><br> | |
− | + | <p><u>Workflow for the screen of a BioBrick library</u></p> | |
− | + | <p>We designed a workflow to build a library, introduce it into UTEX2973 and measure its | |
− | + | characteristics.<br> | |
− | + | ||
− | + | <figure style="text-align:center"> | |
− | + | <img style="height: 400px; width: 600px;" | |
− | + | src="https://static.igem.org/mediawiki/2019/e/e3/T--Marburg--Toolbox_MeasurementWorkflow.svg" | |
− | + | alt="Measurement-workflow"> | |
− | + | <figcaption style="max-width: 2400px; text-align: center"> | |
− | + | Fig.16: Measurement-workflow | |
− | + | </figcaption> | |
− | + | </figure><br> | |
− | + | ||
− | + | </p><br><br> | |
− | + | ||
− | + | <p><u>Testing the reproducibility and standard deviation of the screening workflow</u> | |
− | + | </p> | |
− | + | <p>We tested how reproducible results from our library screening workflow are with a | |
− | + | fluorescence | |
− | + | reporter.<br> | |
− | + | ||
− | + | <figure style="text-align:center"> | |
− | + | <img style="height: 400px; width: 600px;" | |
− | + | src="https://static.igem.org/mediawiki/2019/f/f0/T--Marburg--results_yfp_pam_4787_6_replicates.jpg" | |
− | + | alt="YFP Pam 4787 6 replicates"> | |
− | + | <figcaption style="max-width: 2400px; text-align: center"> | |
− | + | Fig.17: YFP Pam 4787 6 replicates | |
− | + | </figcaption> | |
− | + | </figure><br> | |
− | + | </p><br><br> | |
− | + | <p><u>Application note for the characterization of BioBricks in our chassis</u></p> | |
+ | <p>After calibrating our screening procedure, we decided to share our practical | ||
+ | knowledge | ||
+ | with other end | ||
+ | users.<br> | ||
+ | |||
+ | <a | ||
+ | href="https://static.igem.org/mediawiki/2019/0/07/T--Marburg--Berthold_Application_Note.pdf">Application | ||
+ | Note</a> | ||
+ | </p> | ||
+ | </section> | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
− | + | </section> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</div> | </div> | ||
+ | </div> | ||
</html> | </html> | ||
{{Marburg/footer}} | {{Marburg/footer}} |
Latest revision as of 10:56, 9 December 2019
R E S U L T S
The way to the results we demonstrate here was full of success and failure. Therefore, it was necessary to compare and revise our theoretical plans with the practical work and the associated results. After trying our best to implement our plans, we would like to show you on this page that we have managed to realize some of our goals and are able to show some achievements for every sub-group.
S T R A I N
E N G I N E E R I N G
Strain engineering
Natural competence
Reintroducing natural competence into our S. elongatus strain was an
important goal for us.
To
make sure
that
our S. elongatus UTEX 2973 actually holds the point mutation in the
pilN gene we
thought
it has,
we sequenced
this region - and the results showed the expected mutation
As there were multiple methods at hand that we could use to get our strain
naturally
competent
again,
we tried
all those at hand, making sure we do everything we can to tackle this issue.
Although not our favorite method, we tried integrating an intact copy of the
pilN gene into
neutral site
II of
S. elongatus UTEX 2973 following the example of Li et al., 2018
.
We received pSII-trc-pilN,
the same plasmid used by Li et al., as a gift from Petra Wurmser from the
research group of Prof.
Kaldenhoff in Darmstadt and conjugated it into our strain via triparental
conjugation.
![map](https://static.igem.org/mediawiki/2019/d/d4/T--Marburg--p_pilN-NSII.png)
Beforehand, Petra Wurmser told us that she was not able to successfully
reproduce
the experiment,
motivating
us
to try it ourselves.
We made sure to follow the protocol in the above mentioned paper, transforming
pRL443 and pRL623
into E.
coli
HB101 and pSII-trc-pilN into E. coli HB101. Overnight cultures of these
cells
were inoculated and grown to
OD600≈0.5.
After washing and incubating them together for half an hour, they were mixed
with a
exponentially
growing
S. elongatus culture and incubated for 30 minutes again. Thereafter the
mixture was blotted
on
sterile filters and
incubated on BG11 plates for 24h before being transferred onto BG11 plates
containing kanamycin
(Li et al.,
2018).
It is important to add
that we also went with another approach for conjugation: Martina Carrillo
Camacho
from the working
group of Prof. Dr. Tobias Erb provided us with the pRK2013 plasmid, mentioning
that
she uses it
for conjugation. So we transformed the plasmid into E. coli DH5ɑ and
pSII-trc-pilN into
HB101, performing the
same
procedure with them as stated above.
After a whole week we could actually see growing colonies, though they were only
from attempts
with the
pRK2013 plasmid.
![map](https://static.igem.org/mediawiki/2019/e/e5/T--Marburg--Trafo-pilN-NSII-UDAR.jpg)
We were still excited, directly starting liquid cultures and
running
colony PCRs in hope to find our desired result. Although trying a wide variety
of
primer
combinations,
we were
not able to find any successful integrations, but as the strain was growing on
kanamycin and
colony PCR
does not
always work correctly, we wanted to make sure and tried an actual
transformation: A
YFP construct
was
transformed
into our seemingly competent strain, allowing for easy selection afterwards.
Disappointingly we could not transform the strain. This was in accordance to the
results of
Petra
Wurmser, who
was also not able to reintroduce natural competence into S. elongatus
UTEX
2973 through
this
method.
In hope of better results we decided to try and revert the point mutation in the
pilN
gene with
a CRISPR/Cas12a system.While still working on the system itself as described in
the
![latest conjugation of UTEX2973 with the plasmid pSL2680.](https://static.igem.org/mediawiki/2019/f/ff/T--Marburg--Conjugation_UTEX2973_pSL2680.png)
CRISPR gene editing
Although CRISPR/Cas systems have been discussed as incredibly powerful tools in
genetic
engineering,
they have
not yet been widely used in cyanobacterial research, which is why we set out to
implement such a
system, based
on CRISPR/Cas12a, into our Green
Expansion of the Marburg Collection.
As CRISPR/Cas12a has already been reported to work in S.elongatus UTEX 2973
(Ungerer and Pakrasi, 2016), we
were sure that it could be transformed into a Golden Gate Assembly compatible
version, allowing for more flexible design considerations.
While we started the cloning processes needed to change the existing vector into the
phytobrick
standard, we
tried the vector at hand ourselves, in order to assess its usefulness.
Following the given protocols we constructed a CRISPR/Cas12a vector harboring a
crRNA
and repair
template
designed to revert the point mutation in the pilN gene of our
S.elongatus strain.
After a
few
initial problems we were able to get conjugants and are currently screening for
those
containing
the
desired
edit - more on this approach can be found in the natural competence section of our
results.
In order to modularize this system we built different parts for our genetic toolbox.
First of all
we
created
a
lvl0 part of the Cas12a protein by amplifying the sequence from the pSL2680 plasmid,
including
overhangs
that
enabled us to clone the PCR product into a lvl0 acceptor vector.
Sequencing results proved, that this crucial part was correctly assembled, ready to be used in lvl 1 constructs - which we promptly did, using the following lvl0 parts: pMC0_1_03 + pMC0_2_03 + pMC0_3_07 + pMC0_4_33 + pMC0_5_07 + pMC0_6_17.
![map](https://static.igem.org/mediawiki/2019/a/ae/T--Marburg--Cas12a_CDS_lvl0.png)
The chosen promoter is a rather weak one, so that overproduction of Cas12a is prevented, leading to less toxicity in the cells.
![map](https://static.igem.org/mediawiki/2019/7/74/T--Marburg--Cas12a_CDS_lvl1.png)
Having built this construct, we continued to build the other missing part: the crRNA. The design of the pSL2680 plasmid was mostly kept the same, but in order to have an easy and cheap selection method we switched the lacZ cassette with a GFP cassette.
![crRNA lvl0](https://static.igem.org/mediawiki/2019/7/7e/T--Marburg--Cas12a_crRNA_lvl0.png)
We could show the correct assembly of this part - everything was as we planned in our design meaning that we had all the parts in our MoClo standard.
![[Fig XX seq results of crRNA lvl0],](https://static.igem.org/mediawiki/2019/3/30/T--Marburg--Cas12a_crRNA_lvl0_seq.png)
As the whole system is built for modular cloning in the PhytoBrick syntax, it is
possible to
freely
exchange
the
parts around the Cas12a and crRNA parts. This enables the use of different
promoters, allowing for
easy
screening: Constructs with weaker promoters in front of the Cas12a gene would
lead
to less gene
expression
and
therefore lower toxicity of the whole system. The free exchange of these
promoter
parts can
consequently be
used
for the creation of a library in order to look for the perfectly fitting
promoter
for this system.
Successfully creating these invaluable parts, we were able to establish a
workflow
for faster
cloning in
S.elongatus.
As our system is modularized, it is possible to easily exchange the GFP cassette
for
the desired
crRNA,
which
can be done in a single reaction, further simplifying the cloning process of
CRISPR/Cas12a
constructs.
As shown
before , the cloning process with the pSL2680 can take over a week, is
tedious work and is accompanied by another couple of days waiting for colonies.
In comparison, our system enables for efficient cloning in only four days: On
the first day the construct is assembled in a Golden Gate reaction, which is
thereafter transformed into E.coli. The next day
colonies can be picked,
inoculated and the construct can be
extracted in the evening. On the third day it can be transformed into
S.elongatus -
and on the fourth day colonies can be screened.
The missing piece to apply an edit is the repair template. Skimming through
literature, we
noticed
that
transformation of linear DNA fragments into S.elongatus is supposedly
more efficient
than
the
transformation of whole plasmids (Almeida et
al., 2017) - and we were able to verify this fact in our own
experiments. This further simplifies our above mentioned workflow, as we are
able
to simply PCR the needed repair template from a DNA sequence and use the PCR
product for
transformation into
S.elongatus. Our toolbox has a
special feature that can be used for exactly this workflow: a NotI cutting
site
can be found
in
our
constructs,
which is used to linearize them, so that they can be more efficiently
transformed.
We are more than certain that our modular CRISPR/Cas12a proves to be an
invaluable
contribution
to the
tools
available in cyanobacterial research, especially for the Golden Gate
community,
which is
growing
bigger
and
bigger every year - also thanks to the iGEM headquarters finally integrating
the
TypeIIS
standard into
the
competition!
Cyanobacterial shuttle vectors
As we have already clarified in the description part, self replicating shuttle vectors are essential for many workflows, as the gene expression levels are higher and non of the tedious selection processes that come with genomic integrations have to be done.
On our road to the modular vector we were seeking, we firstly cured our own S. elongatus UTEX 2973 strain of its pANS plasmid. This was done by transforming the pAM4787 vector, which holds a spectinomycin resistance as well as a YFP cassette (Chen et al., 2016). Due to plasmid incompatibility - explained here in our design section - and because antibiotic pressure is applied, the pANS plasmid was over time cured from the strain, which then just kept the pAM4787 plasmid. Transformation was done by conjugation with the pRK2013 plasmid in DH5ɑ and the pAM4787 in HB101. Both were grown to an OD600≈0.5, washed in LB and mixed with S. elongatus which was grown to late exponential phase and then washed in BG11. We could clearly show, that the conjugant strain bears the pAM4787 plasmid if selective pressure is held up.
![https://static.igem.org/mediawiki/2019/e/eb/T--Marburg--YFPconstructConjugantFACS.png](https://static.igem.org/mediawiki/2019/e/eb/T--Marburg--YFPconstructConjugantFACS.png)
This was followed by us starting to culture the pAM4787 bearing strain without antibiotics again, slowly removing selective pressure from the cells. As the plasmid does not give them any other advantage and is probably just more metabolic burden due to the constantly produced YFP proteins it is slowly being lost. We could prove this in multiple setups: with the flow cytometry device we were kindly granted access to we could clearly show the missing YFP signal in the cured S. elongatus strain and logically this could also be observed over our UV table.
![https://static.igem.org/mediawiki/2019/c/c4/T--Marburg--CuredStrainsFACS.png](https://static.igem.org/mediawiki/2019/c/c4/T--Marburg--CuredStrainsFACS.png)
![https://static.igem.org/mediawiki/2019/d/da/T--Marburg--CuredStrainUV.png](https://static.igem.org/mediawiki/2019/d/da/T--Marburg--CuredStrainUV.png)
Furthermore we performed colony PCRs as a test. We sent our plasmid-free strain to Next Generation Sequencing in order to ensure that the strain really has lost the pANS plasmid.
![gel pcr](https://static.igem.org/mediawiki/2019/e/e9/T--Marburg--ColonyPCRcuredStrain.jpg)
Our next step was the characterization of the cyanobacterial shuttle vector mentioned in our design section. In an extensive flow cytometry experiment we assessed the fluorescence of a transformed YFP-construct in our cured strain, showing that the shuttle vector with the minimal replication element can be maintained in S. elongatus UTEX 2973.
After another four weeks of cultivation we looked at our cultures again on the UV table to check if fluorescence was still present and the high intensity of fluorescence proved to us, that the plasmid is still stably replicated in our strain, showing us, that the minimal replication element does indeed work in our strain. For further analysis we performed qPCR with this transformed strain, in order to check the copy number of the vector. We used the copy number of pANL as a reference, which is supposedly at ~2,6 copies per chromosome (Chen et al., 2016). Our data shows a ~4,5 times higher copy number relative to pANL, meaning that the construct is maintained with approximately 11,7 copies per chromosome.
For further analysis of this part (BBa_K3228069) we performed a Quantitative-Polymerase-Chain-Reaction (qPCR) with this transformed strain, in order to check the copy number of the vector. This proved to be difficult in Cyanobacteria, due to variation in genome copy number (Griese et al., 2011; Chen et al., 2012). To overcome this problem, we noticed that the copynumber of pANL stays rather stable with 2,6 copies per chromosome (Chen et al., 2016). Therefore we performed the qPCR with pANL as an additional target. For each target, we chose three different primer pairs, with known efficiency. Five technical and two biological replicates were used. The samples were normalised to the chromosome and pANL was used to identify the total copy number per chromosome (figure 14). Our data shows a ~4,5 times higher copy number relative to pANL, meaning that the construct is maintained with approximately 11,7 copies per chromosome. This is comparable with the copynumber of pANS in Chen et al. 2016.
![Copynumber Evaluation of Ori-part via qPCR](https://static.igem.org/mediawiki/2019/d/d6/T--Marburg--Parts--qPCR-Lvl1.png)
Additionally we measured the fluorescence signals in a plate reader at different optical densities and could again confirm high fluorescence signals, indicating strong gene expression in constructs built around this replication element.
![diagramm](https://static.igem.org/mediawiki/2019/f/f0/T--Marburg--results_yfp_pam_4787_6_replicates.jpg)
All this data confirms that the construct actually works and can be reliably used as a cyanobacterial shuttle vector, proving that BBa_K3228069 works as intended, thus functioning as our validated part. This assumption is solidified by all our sequence data, showing that the shuttle vectors were completely assembled as planned in our design section.
M A R B U R G
C O L L E C T I O N 2.0
Marburg Collection 2.0
Overview over the expansion of the Marburg Collection:
We added 55 new parts to the Marburg Collection, adding several new features such as the Green expansion, including a kit for the Modularized Engineering of Genome Areas (M.E.G.A.) and the first MoClo compatible shuttle vector for cyanobacteria. Additionally, we offer a set of reporters suitable for characterization of BioBricks in cyanobacteria and ribozymes for a more stable and species independent transcription. We also provide standardized measurement vectors that were generated using our designed placeholders.
Overview over the different expansions in the Marburg Collection 2.0
To give a better overview we show here the different expansions we added to the
Marburg
Collection:
Parts
Sequencing results of the lvl0 parts
We built and validated 55 new BioBricks this year. They are all listed in the
Registry of
Standard
Biological Parts
(Part range BBa_3228000 to BBa_32280103). All lvl0 Parts were validated by complete
sequencing.
Building constructs to test the lethality of origin of transfer
If plasmids reach a certain size normal transformation protocols are not feasible
anymore
to bring
the
plasmid into
the host.
For the transformation of such huge megaplasmids we designed an “origin of transfer”
BioBrick that
makes
it possible
to directly transport plasmids of any size from one species to another. To test if
this
sequence would
result in any
toxicity in a genomic context (source things where genome parts can be exchanged by
integrating such
sequences) we built
it into an integration vector.
Sequencing results of the lvl1 parts for modularized genome integrations
We successfully build two integration cassettes from our rationally designed
artificial
neutral
integration sites
(a.N.S.o. 1 and 2) and verified them by sequencing. These parts contained the
“origin of
transfer” to
test
their lethality
in the aforementioned experiment.
Workflow to integrate a modularized integration cassette
We established a workflow on how to integrate a cassette - from lvl0 Parts to a finished change in genome. With UTEX 2973 this is possible in less than five days, while in PCC7942 the same integration would take a whole month.
Using the placeholder to build standard measurement vectors
We successfully used our placeholders to build and validate the standardized
measurement
vectors for
promoters,
ribosomal binding sites and coding sequences. We evaluated the cost and time savings
from a library
assembly with a
sample size of 25.
Through our design decision to build placeholders we managed to cut the workload for
a
high throughput
assembly by around 72%
and the invested financial resources by 40 % with just a sample size of 25
assemblies.
Workload for placeholder
Task | Time per sample [min] | Samples | Total |
---|---|---|---|
Assembly of entry vector (7 part assembly) | 1 | ||
Golden Gate reaction | 20 | 1 | 20 |
Transformation | 15 | 1 | 15 |
Preparing 10 overnight cultures + purification | 15 | 10 | 150 |
Test digesting | 5 | 5 | 25 |
Inserting Promoters (2 part assembly) (25 BioBricks) | 25 | ||
Golden Gate reactions (simplified with a mastermix) | 5 | 25 | 125 |
Transformation | 10 | 25 | 250 |
Preparing overnight cultures + purification | 15 | 50 | 750 |
Total | 1335 | ||
In hours | 22,25 | ||
Workflow without placeholders | |||
lvl1 Golden Gate Assembly | 25 | ||
Golden Gate reaction | 5 | 25 | 125 |
Transformation | 10 | 25 | 250 |
Preparing 10 overnight cultures + purification | 15 | 250 | 3750 |
Test digesting | 5 | 125 | 625 |
Total | 4750 | ||
In hours | 79,16666667 | ||
Differences in hours | 56,91666667 | ||
Percentage saved | 0,72 |
Cost for placeholder
Major cost point | Price [€] | Samples | Total |
---|---|---|---|
With placeholder | |||
lvl1 Golden Gate mix for measurement vector assembly | 1 | ||
BsaI | 2 | 1 | 2 |
BsmbI | 2 | 1 | 2 |
Plasmid purification | 2 | 5 | 10 |
Test digesting | 5 | ||
Enzyme | 1 | 5 | 5 |
Aggarose gel and ladder (approximation) | 0,5 | 2,5 | 1,25 |
Sequencing (estimated) | 3 | 3 | 9 |
lvl1 Golden Gate mix for inserting the promoter> | 25 | ||
BsaI | 2 | 25 | 50 |
BsmbI | 2 | 25 | 50 |
Plasmid purification | 2 | 50 | 100 |
Sequencing | 3 | 50 | 150 |
Total | 378,25 | ||
Without placeholder | |||
lvl1 Golden Gate mix for measurement vector assembly | 25 | ||
BsaI | 2 | 25 | 50 |
BsmbI | 2 | 25 | 50 |
Plasmid purification | 2 | 125 | 250 |
Test digesting | 125 | ||
Enzyme | 1 | 125 | 125 |
Aggarose gel and ladder (approximation) | 0,5 | 62,5 | 31,25 |
Sequencing (estimated) | 3 | 41,625 | 124,875 |
Total | 378,25 | ||
Difference | 251,875 | ||
Percentage saved | 0,4 |
Construction of a promoter library with standard measuring vectors
We built a promoter library using our standard promoter measurement vector and 25
BioBricks.
Here we show a list of all the BioBricks we used.
Workflow for the screen of a BioBrick library
We designed a workflow to build a library, introduce it into UTEX2973 and measure its
characteristics.
Testing the reproducibility and standard deviation of the screening workflow
We tested how reproducible results from our library screening workflow are with a
fluorescence
reporter.
Application note for the characterization of BioBricks in our chassis
After calibrating our screening procedure, we decided to share our practical
knowledge
with other end
users.
Application
Note