(Foundational experiments with Synechococcus elongatus UTEX 2973)
Abstract?
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At this passage we show how to do genetic modifications to regain the natural competence of Synechococcus elongatus UTEX 2973. With these methods we succeed the natural transformation of plasmids in UTEX2973.
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
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.
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 [Link to design of Natural Competence], 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!
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We were able to construct and test XXX different parts in S. elongatus. Further we were able to test the different constructs (Promotors, RBS, CDS, Terminators) for their efficiency by measuring the fluorescence of the cultures through the expressed YFP-gene (Platereader and FACS, link?).
Results of the 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:
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 2 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 UTEX2973 this is possible in less than 5 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.
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. The exact procedures and a discussion of the results in regards to its reproducibility can be found in (Link to measurement)
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
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With our engineered strain and developed toolbox we aim to produce a viable alternative to kerosene by fixing CO2 and convert it into chemical resources. We decided on the bio jet fuel AMJ-700t, which consists of limonene (50%), farnesene (40%) and p-cymene (10%).