Our Parts

Our contribution to the iGEM community is a collection of seven new BioBricks we created for our project.

Best Basic Part
BBa_K2203000 - GamS protein

We have chosen to nominate GamS as our central and best part. It is an indispensable component in any cell-free protein synthesis system. We are happy to introduce it to the iGEM community and are certain it will be highly valuable to future teams working in a cell-free environment.

GamS protects linear double-stranded DNA templates from degradation by exonucleases in cell-free reactions¹. Adding GamS in any form to a cell-free reaction will increase protein expression two to threefold. To see our results demonstrating this effect, click here

Best Composite Part
BBa_K2203001 - T7-GamS 6X His-Tag

This composite part is a version of GamS optimized for expression in bacteria. The T7 promoter will make highly efficient GamS transcription possible in any lysate or bacteria that contain the T7 RNA polymerase. A practical way to use GamS is to transform bacteria with this plasmid and then make lysate from them. That way, all reactions based on this lysate will already contain enough GamS to ensure high protein expression.

On top of that, this part is equipped with a 6X His-Tag. With this tag, a simple affinity purification can be performed to recover the expressed GamS.

BBa_K2203002 - T7-LacZalpha

We improved the basic part LacZalpha (BBa_I732006) with a T7 promoter to make it functional in the cell-free environment of our E.Coli lysates. This is our Gold Requirement.

Our main reporter gene for this project is LacZalpha. LacZalpha makes up the first 220 nucleotides of the LacZ gene. Most E.Coli strains express LacZ constitutively, however some specially engineered strains have a mutation that deletes the sequence coding for LacZalpha. We work with those mutated strains mostly.
Expressed LacZalpha will assemble with the rest of the translated LacZ to form a functional beta-galactosidase enzyme. If we express LacZalpha in a lysate from cells having the mutation, then they will end up having the complete enzyme in the solution. We then measure the presence of this enzyme by observing its activity. The enzyme will cleave a yellow substrate (Chlorophenol Red-β-D-galactopyranoside) and make it purple in the process. This colour change is strong enough to be visible to the naked eye, and it is the basis of our detection scheme.

The T7 promoter in front allows for swift and efficient expression by a T7 RNA polymerase.

BBa_K2203003 - HCV Toehold A LacZalpha

This composite part is a toehold switch specific to a sequence from the Hepatitis C virus, genotype 1a. The toehold switch is made up of a toehold domain and followed by the coding sequence of a reporter gene. Once the toehold switch is expressed, the toehold domain will curl up into a hairpin structure and prohibit translation of the reporter.
In order to translate the reporter gene, the toehold first needs to unfold. This happens only if a trigger is present. A trigger sequence is complementary to the toehold domain and will thus bind to the switch. Upon binding, the toehold unfolds.

This particular toehold was found using our software, Toehold Designer. We show that our software outputs the expected result since this toehold works fine with our lysates and provide the iGEM community with a new way of detecting Hepatitis C.

BBa_K2203004 - Zika Toehold 27B LacZalpha

First documented in a paper by Pardee et al², we base our early results on this toehold switch. It gets triggered by a complementary sequence unique to the Zika virus and is thus used to diagnose infection with Zika.

BBa_K2203005 - Zika Toehold 32B LacZalpha

Similarly this composite part too comes from the paper by Pardee et al² and was tested successfully in our cell-free environment. It's a toehold switch that recognizes another unique sequence of the Zika genome.

BBa_K2203006 - HCV Toehold B LacZalpha

This Hepatitis C toehold switch was also generated by our software Toehold Designer. It again confirms that our software produces reliable and solid results.

References

1. Murphy, Kenan C. "The λ Gam protein inhibits RecBCD binding to dsDNA ends." Journal of molecular biology 371.1 (2007): 19-24.

2. Pardee, Keith, et al. "Rapid, low-cost detection of Zika virus using programmable biomolecular components." Cell 165.5 (2016): 1255-1266.

Team:EPFL/Parts