Team:INSA-Lyon/Composite Part
Summary
The aim of our project is to create a device to detect Sexually Transmitted Infections (STIs). To that end, we have created three series of BioBricks that enabled us to investigate and characterize the function of different pieces of our self-test.
One of the targets: HIV reverse transcriptase
The objective of our project is to detect some Sexually Transmitted Infections (STIs) and especially, HIV with the HIV reverse transcriptase and HBV with its surface antigen HBsAg. In order to test our detection device, we created two composite parts which contain the coding sequences of p51 and p66 subunits of HIV reverse transcriptase respectively.
BBa_K1934060: p51 subunit of HIV reverse transcriptase.
This composite part contains the coding sequence of the p51 subunit (UniProtKB P03366, PDB 5D3G_B), which directly comes from the genome of the HIV 1 group M subtype B, under the control of T7 promoter, a RBS and a T7 terminator.
BBa_K1934061: p66 subunit of HIV reverse transcriptase.
This composite part contains the coding sequence of the p66 subunit (UniProtKB P03366, PDB 5D3G_A), which directly comes from the genome of the HIV 1 group M subtype B, under the control of T7 promoter, a RBS and a T7 terminator.
The anchor: streptavidin chimeric protein
As an anchor, we chose to use the streptavidin protein combined with one or two cellulose binding domain(s) in order to be easily fixed onto a cellulose paper.
BBa_K1934020: Streptavidin with Cellulose Binding Domains (CBDs).
This composite part contains the coding sequence of the streptavidin protein combined with two CBDs, which corresponds to the Stanford-Brown 2014 iGEM team part (BBa_K1499004) , under the control of a pTAC promoter, a strong RBS and a bidirectional terminator.
BBa_K1934000: RFP with Cellulose Binding Domains (CBDs)
To verify the efficacy of the CBDs for binding to the cellulose, we created this composite part in which the coding sequences for two different CBDs were fused respectively upstream and downstream of the coding sequence for RFP.
BBa_K1934030: Streptavidin with CBD-CipA (Cellulosomal scaffolding protein A)
In order to test another type of CBDs, we created this composite part which presents the coding sequence of streptavidin followed by the coding sequence of CBD-CipA (Cellulosomal scaffolding protein A) protein. As CBD-CipA shows a better interaction with cellulose, this part is an improvement of the streptavidin with classical CBDs (BBa_K1499004). We have submitted this part to the “Best new composite part” special prize.
Characterization of pTAC promoter
pTAC is a promoter that we used in our different composite parts (eg for surproduction of proteins). As it’s induced by IPTG, by linking a reporter gene downstream of pTAC, the two following parts allowed us to characterize the promoter's responsiveness to IPTG, to quantify the promoter leakage and thus to improve the basic part containing the pTAC promoter sequence (BBa_K864400).
BBa_K1934040: RFP under control of pTAC promoter
This composite part contains the coding sequence of the RFP reporter gene downstream of pTAC and strong RBS sequences.
BBa_K1934050: CFP under control of pTAC promoter.
This composite part contains the coding sequence of the CFP reporter gene downstream of pTAC and strong RBS sequences.
