Team:Marburg/Demonstrate
D E M O N S T R A T E
Strain Engineering
“Started from the bottom, now we're here” - Drake
Our project has three goals. First, we want to establish the cyanobacteria Synechococcus elongatus UTEX 2973 and restore its natural competence. Secondly, we wanted to show the best growing conditions with the goal of standardization for working with UTEX 2973. The final goal is to create a suitable Golden Gate toolbox for our strain and share it with other iGEM teams and researchers.
In our iGEM year we worked to restore the natural competence of Synechococcus elongatus UTEX 2973, analyzed different cultivation parameters to accelerate the growth speed including finding the best conditions. Besides that we expanded the Marburg Collection from 2018 and are able to deliver a toolbox of 55 parts with free access to the world. On top of that, we automated plasmid purification and colony picking via the Opentrons OT-2.
Toolbox
"Great things are not done by impulse, but by a series of small things brought together." – Vincent van Gogh
This year we expanded the Marburg Collection from 2018 with 55 new parts to the Marburg Collection
2.0.
With our developed workflow we could characterize our parts and
compare them with a second measurement
method: FACS/flow cytometry. We added two new features for genome engineering of
cyanobacteria: A CRISPR/Cas12a
guided
knockout
system as well as a
modularized assembly of repair templates for the knock in of genes
(M.E.G.A. expansion).
This includes
integration sites that target conventional neutral sites in cyanobacteria but we also rationally
designed two novel integration sites based on RNA-seq data. Additionally, we offer the first MoClo
compatible shuttle vector for cyanobacteria and
characterized gene expression based on that origin of replication.
We used our new shuttle vector
to
build standardized devices for the characterization of BioBricks in cyanobacterial chassis to improve
the reproducibility of results and to simplify large scale assemblies. For this we used placeholders, a
novel part type that aids in the construction of a larger set of parts by reducing the involved cost
and
workload significantly. Additionally, we tested our toolbox with PCC 7942 to show that the Marburg
Collection 2.0 is also working with similar cyanobacteria.
We offer free access to the data of
our
characterization, enabling the iGEM community and scientists to choose the parts based on this data.
To
improve the measurement method applicable for cyanobacteria we focused on measurements via
luminescence
reporters over fluorescence reporters, because of the fact that cyanobacteria emit autofluorescence.
This way our results are way more accurate, because of the reduced background noise. The higher
accuracy
is obviously visible during the measurement of our parts, where we could see a difference of
5x105
between the background noise and the signal, which implements that already a small amount of sample
has
a more intensive signal.
Hereby, we want to encourage the community of young scientists to work with the fastest phototrophic organism Synechococcus elongatus UTEX 2973 because of its high relevance for biotechnological applications.
Automation
We implemented a completely automated cloning workflow in the Opentrons OT-2. We have published our complete work and can now provide you with the soft- and hardware to bring you the fully automated Colony Picking Unit C.P.U. as well as the plasmid purification protocol. We made as fully accessible and cheap as possible to give access to as many people as possible.