Difference between revisions of "Team:Marburg/Results"
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href=https://doi.org/10.1016/j.ymben.2018.06.002> Li et al., 2018 </a> . We received pSII-trc-pilN, | href=https://doi.org/10.1016/j.ymben.2018.06.002> Li et al., 2018 </a> . 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. | 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. <figure | + | Kaldenhoff in Darmstadt and conjugated it into our strain via triparental conjugation.</p> <figure |
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| − | CRISPR gene editing | + | <u>CRISPR gene editing</u> |
<p>CRISPR gene editing | <p>CRISPR gene editing | ||
Although CRISPR/Cas systems have been discussed as incredibly powerful tools in genetic engineering, | Although CRISPR/Cas systems have been discussed as incredibly powerful tools in genetic engineering, | ||
Revision as of 17:16, 8 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.
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 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
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 right, 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. 4 Bilder hiernoch rein ! 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/Cas system.While still working on the system itself as described in the design section [links to design], we used the pSL2680 plasmid Ungerer and Pakrasi, 2016 to engineer our strain. We followed their protocol (available here on Addgene) to construct a guiding crRNA, leading the Cas12a enzyme to the mutated pilN 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 .[Fig.XX Seq results nicht da ?] 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 Synechococcus elongatus strain. We contacted Prof. Dr. James W. Golden from the University of California, San Diego to ask for advice [link to golden skype call] 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 Na2S2O3 (sodium thiosulfate) in their BG11 plates and we could find literature to support this advice (Thiel et al., 1989). 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 H2O2 can be formed, which can drastically hinder bacterial growth (Tanaka et al., 2014). The thiosulfate from Na2S2O3 is oxidized as H2O2 is reduced to 2 H2O - 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 ( Ungerer and Pakrasi, 2016 , Yu et al., 2015 , Wendt et al., 2016 ). We are currently screening for correct edits and are certain to hold a naturally competent strain in our hands in a matter of days [Fig.XX new plates from Vincas conj].
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 [Link to
MarburgCollection].
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 [Link to Design of CRISPR ].
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
lvl 0 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 lvl 0 acceptor vector
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
![m[Fig XX plasmid map of crRNA lvl0]. Through sequencingap](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
[Link to design of CRISPR] 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 [Fig XX linear
transformation plate pic]. 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 [Link to shuttle vector design] - 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.
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.
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.
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 inS. 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 the 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 Primerpiars, with known Efficiency. Five technical and two biological replicates were used. The samples were normalised to the genome and pANL were used to identify the total copy number per Genome (figure xx). 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 in compatible with the copynumber of pANS in Chen et al. 2016.
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.
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 [Link to design of shuttle vectors] .
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 LVL 0 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 LVL 0 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. For sequencing results see the dropdown menu below.
Sequencing results of the LVL 1 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 LVL 0 Parts to a finished change in genome. With UTEX 2973 this is possible in less than five days, while in PCC 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 | |||
| LVL 1 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 | |||
| LVL 1 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 |
| LVL 1 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 | |||
| LVL 1 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