Difference between revisions of "Team:Toulouse/Parts"

In the list below you will find an overview over the BioBrick parts we added to the registry, which were created by the iGEM Toulouse 2015 team.

For our project, we worked on three different modules : attract the varroa, kill the mite and finally the circadian switch to alternatively produce the two molecules of interest, butyrate during the day and formate during the night.

The chassis we used is Escherichia coli, and this bacterium is not able to naturally produce butyrate. That is why we introduced genes from others bacterial strains to synthesize this molecule.

Gene from Escherichia coli involved in the butyrate pathway that enables its production directly from acyl-coAs. This group of enzymes catalyzes the hydrolysis of acyl-CoAs into free fatty acid (in our case, butyryl-coA into butyrate) plus reduced coenzyme A (CoA-SH).

Gene from Clostridium acetobutylicum was introduced in our bacterium after codon optimization in order to obtain a better expression in E. coli. The crt enzyme substrate is 3-hydroxybutyryl CoA, and the product is Crotonyl CoA. This reaction does not need any coenzyme.

This BioBrick construction is composed of a constitutive promoter p(Bla) (BBa_I14018) and 5 genes from three different micro-organisms : in yellow are the E.coli genes, in blue those from Clostridium acetobutylicum and finally, the purple gene is from Streptomyces collinus. A Ribosome Binding Site (RBS) represented by a green circle (BBa_B0030), is added between each gene in order to improve the proteic synthesis. Finally, a strong terminator (BBa_B1006) represents the end of the sequence.

tesB and crt have been described previously. ccr encodes crotonyl CoA reductase, an oxidoreductase which acts on the double bond CH=CH. hbd in Clostridium acetobutylicum encodes 3-hydroxybutyryl-CoA dehydrogenase, an oxidoreductase which catalyses the formation of alcohol function. atoB, in E.coli, encodes acetyl CoA acetyltransferase which catalyses the condensation of two acetyl CoA.

This BioBrick construction is the same as previously, but does not contain the ccr gene from Streptomyces collinus. It is composed of a constitutive promoter p(Bla) (BBa_I14018) and of 4 genes from two different micro-organisms : in yellow are the genes from E. coli, and in blue those from Clostridium acetobutylicum. The green circles correspond to the strong RBS (BBa_B0030) sequences based on Ron Weiss thesis and the red one is the terminator (BBa_B1006).

To obtain the second module, we decided to produce formic acid. Indeed, this molecule has two benefits. The first is that the acaricide effect has been demonstrated, and the second is the natural production of the compound by E. coli, the bacterium we chose as chassis. Glucose is the initial substrate and it is degraded into pyruvate during glycolysis. Finally, formate is synthesized thanks to two key genes : pflA and pflB.

pflB encodes the pyruvate formate lyase, an enzyme which catalyses the cutting between C1 and C2 carbons of pyruvate. This enzyme is oxygen-sensitive and is only active in microaerobic or anaerobic conditions.

pflA encodes the pyruvate formate lyase activase, an enzyme which activates pflB.

The formate compound is naturally produced by E. coli, that is why we decided to overexpress the two essential genes.

To test the formate production, pflB and pflA are put together with two RBS (BBa_B0030) in front of them to improve the proteic synthesis. A strong terminator (BBa_B1006) ends the sequence.

As said previously, we decided to produce butyric acid in our trap during the day and formic acid to kill the mite during the night. We designed a light response system which is improved to obtain an on/off switch of genic expression. We adapted this system because we did not want to obtain only an on/off switch but a switch of genic expression between two different polycistronic genes following the presence or the absence of light.

The center of the light sensor is composed of membrane proteins PCB (chromophore phycocyanobilin) and the hybrid protein Cph8 (EnvZ and Cph1).

PCB protein comes from a cyanobacterium Synechocystis sp PCC 6803 and to be synthesized, it needs the expression of two genes: heme oxygenase (Ho1) and biliverdin reductase (PcyA).

Gene required for chromophore synthesis in photosynthetic light-harvesting complexes, photoreceptors, and circadian clocks. ho1, along with pcyA, converts heme into the chromophore phycocyanobillin (PCB).

The biobrick is composed of a strong RBS (BBa_B0030) and Ho1 coding region (BBa_K566022).

Gene required for chromophore synthesis in photosynthetic light-harvesting complexes, photoreceptors, and circadian clocks.

Biobrick composed of a strong RBS (BBa_B0030) and pcyA coding region (BBa_K566023).

This biobrick contains OmpC promoter (BBa_R0082), a lacI box separated from cI gene by a RBS sequence (BBa_B0030).

Strong promoter BBa_J23119. Strong RBS BBa_B0030 Chimeric Red light receptor cph8 BBa_I15010. It is a chimeric Red light receptor cph8 genetic construction. This chimeric Red light receptor cph8 (cph1-envZ) is inactivated by phycobilin PCB photosynthetic light-harvesting, preventing the phosphorylation of ompR

Uses a positively regulated promoter (P_ompC) and a lacI binding site to regulate the expression of cI. This construction was designed to be regulated by cph8 red light receptor (cph1-envZ chimeric protein). The purpose was to create a circadian switch to alternate the expression of two sets of genes (formate/butyrate).