Partnership

Collaboration with Tel-Aviv University IGEM STAUbility Team

The purpose of the collaboration: to provide a solution to the genomic instability problem of the plasmid. When a genetic segment is inserted into a host bacterium, there is a genomic instability of the plasmid (the instability is cause by the fact that the plasmid is a foreign segment in the bacterium). The instability can cause low expression of the plasmid genes or even in the loss of the same insert after several generations due to random mutation. Tel-Aviv IGEM STAUbility team has proposed a way to reduce the genomic instability problem of plasmid by making various sequential changes.

Plasmid Components (before any change - this is the plasmid that was described to the Tel-Aviv IGEM STAUbility team):
Leader (signal peptide)
Passenger genes:

6xHIS tag
CBD: Cellulose Binding Domain
Endo5a - Cellulase enzyme sequence
GS linker (between CBD & Endo5a)
Peptide Linker (between Endo5a & AIDA-I b-barrel)
Beta barrel
Back-bone (including origin of replication (ORI), ampicillin resistance (amp)

Figure 1- Plasmid map given to IGEM Tel-Aviv University STAUbility team.

Our request from IGEM Tel-Aviv STAUbility Team was to maintain the following areas in the plasmid (in order not to compromise plasmid expression):

The origin of replication should be maintained in its nucleotide sequence.
ORFs: HIS, CBD, GS, Endo5a, AIDA-I: (3549-588, refers to positions on the plasmid. See Figure 1) forward. Should be maintained in its amino acid sequence level.
Amp resistance marker: (2317-3177, refers to the position on the plasmid. Please see Figure 1 for location) reverse strand. Preserved in its amino acid sequence level.
Amp promoter: (3219-3247, refers to position on the plasmid. please see Figure 1 for location) reverse strand. Maintained in its nucleotide sequence level.
Promoter for 6xHIS tag, CBD, GS linker, Endo5a AIDA-I (3248-3548, refers to position on the plasmid. Please see Figure 1 for location)- maintained in its nucleotide sequence level.

The plasmid stability was optimized by the IGEM Tel-Aviv STAUbility Team according to the following parameters:

Minimization of GC content on the plasmid where applicable - High percentage of GC content may impair the genomic stability of the plasmid [1].
Codon Usage - Adapt the use of the codons to the use of the host which is E. Coli in our case.
Slippage-site-regions (SSR) – SSRs - are sites of repeating short sequences. These sequences can potentially cause polymerase slippage. Elimination of slippage-site regions may reduce genomic instability [2].

Results- the resulting plasmid map as was planned by the Tel-Aviv IGEM STAUbility team:
partnership image two

Figure 2- Optimized plasmid map by the Tel-Aviv IGEM STAUbility team.

The evolutionary failure mode (EFM) calculator:
Evolutionary Failure Mode (EFM) Calculator – is a calculator that computationally detects common sources of genetic instability in an input DNA sequence [3]. We used the calculator to evaluate the changes made by Tel-Aviv IGEM STAUbility team to improve the stability of the plasmid. Note: a lower score given by the calculator means higher stability of the DNA sequence.

Before optimization
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after optimization
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Figure 3- Plasmid EFM scores before and after optimization

In conclusion: It can be clearly seen from the EFM simulations shown in Figure 3 that there has been an increase in the genomic stability of the plasmid upon the modification performed by the Tel-Aviv team on our plasmids.
We would like to gratefully acknowledge Tel Aviv University’s IGEM team of 2020 for this fruitful collaboration.

[1] Kiktev, D. A., Sheng, Z., Lobachev, K. S., & Petes, T. D. (2018). GC content elevates mutation and recombination rates in the yeast Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences, 115(30), E7109-E7118.

[2] Viguera, E., Canceill, D., & Ehrlich, S. D. (2001). Replication slippage involves DNA polymerase pausing and dissociation. The EMBO journal, 20(10), 2587–2595.

[3] Jack, B. R., Leonard, S. P., Mishler, D. M., Renda, B. A., Leon, D., Suárez, G. A., & Barrick, J. E. (2015). Predicting the genetic stability of engineered DNA sequences with the EFM calculator. ACS synthetic biology, 4(8), 939-943.

Team:BGU-Israel/Partnership

Partnership

Collaboration with Tel-Aviv University IGEM STAUbility Team