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
Reintroducing natural competence into our S. elongatus strain was an
important goal for us.
our S. elongatus UTEX 2973 actually holds the point mutation in the
pilN gene we
this region - and the results showed the expected mutation
As there were multiple methods at hand that we could use to get our strain
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
to try it ourselves.
We made sure to follow the protocol in the above mentioned paper, transforming
pRL443 and pRL623
HB101 and pSII-trc-pilN into E. coli HB101. Overnight cultures of these
were inoculated and grown to
After washing and incubating them together for half an hour, they were mixed
S. elongatus culture and incubated for 30 minutes again. Thereafter the
mixture was blotted
sterile filters and
incubated on BG11 plates for 24h before being transferred onto BG11 plates
(Li et al.,
It is important to add
that we also went with another approach for conjugation: Martina Carrillo
from the working
group of Prof. Dr. Tobias Erb provided us with the pRK2013 plasmid, mentioning
she uses it
for conjugation. So we transformed the plasmid into E. coli DH5ɑ and
HB101, performing the
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
colony PCRs in hope to find our desired result. Although trying a wide variety
not able to find any successful integrations, but as the strain was growing on
always work correctly, we wanted to make sure and tried an actual
into our seemingly competent strain, allowing for easy selection afterwards.
Disappointingly we could not transform the strain. This was in accordance to the
was also not able to reintroduce natural competence into S. elongatus
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
CRISPR gene editing
Although CRISPR/Cas systems have been discussed as incredibly powerful tools in
not yet been widely used in cyanobacterial research, which is why we set out to
implement such a
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.
The chosen promoter is a rather weak one, so that overproduction of Cas12a is prevented, leading to less toxicity in the cells.
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.
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.
As the whole system is built for modular cloning in the PhytoBrick syntax, it is
parts around the Cas12a and crRNA parts. This enables the use of different
promoters, allowing for
screening: Constructs with weaker promoters in front of the Cas12a gene would
to less gene
therefore lower toxicity of the whole system. The free exchange of these
for the creation of a library in order to look for the perfectly fitting
for this system.
Successfully creating these invaluable parts, we were able to establish a
As our system is modularized, it is possible to easily exchange the GFP cassette
can be done in a single reaction, further simplifying the cloning process of
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
and on the fourth day colonies can be screened.
The missing piece to apply an edit is the repair template. Skimming through
transformation of linear DNA fragments into S.elongatus is supposedly
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
to simply PCR the needed repair template from a DNA sequence and use the PCR
S.elongatus. Our toolbox has a
special feature that can be used for exactly this workflow: a NotI cutting
can be found
which is used to linearize them, so that they can be more efficiently
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.
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 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.
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.
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
Sequencing results of the lvl0 parts
We built and validated 55 new BioBricks this year. They are all listed in the
(Part range BBa_3228000 to BBa_32280103). All lvl0 Parts were validated by complete
Building constructs to test the lethality of origin of transfer
If plasmids reach a certain size normal transformation protocols are not feasible
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
(a.N.S.o. 1 and 2) and verified them by sequencing. These parts contained the
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
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|
|Preparing 10 overnight cultures + purification||15||10||150|
|Inserting Promoters (2 part assembly) (25 BioBricks)||25|
|Golden Gate reactions (simplified with a mastermix)||5||25||125|
|Preparing overnight cultures + purification||15||50||750|
|Workflow without placeholders|
|lvl1 Golden Gate Assembly||25|
|Golden Gate reaction||5||25||125|
|Preparing 10 overnight cultures + purification||15||250||3750|
|Differences in hours||56,91666667|
Cost for placeholder
|Major cost point||Price [€]||Samples||Total|
|lvl1 Golden Gate mix for measurement vector assembly||1|
|Aggarose gel and ladder (approximation)||0,5||2,5||1,25|
|lvl1 Golden Gate mix for inserting the promoter>||25|
|lvl1 Golden Gate mix for measurement vector assembly||25|
|Aggarose gel and ladder (approximation)||0,5||62,5||31,25|
Construction of a promoter library with standard measuring vectors
We built a promoter library using our standard promoter measurement vector and 25
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
Testing the reproducibility and standard deviation of the screening workflow
We tested how reproducible results from our library screening workflow are with a
Application note for the characterization of BioBricks in our chassis
After calibrating our screening procedure, we decided to share our practical
with other end