Team:Toulouse/Parts
NE PAS MODIFIER PROUT
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.
| Name | Type | Genic construction | Lenghth (bp) | References |
|---|---|---|---|---|
| BBa_K1587000 | Composite (with RBS) | RBS-ho1 | 744 | [13] |
| BBa_K1587001 | Basic part | tesB | 861 | [3][4][5] |
| BBa_K1587002 | Composite (with RBS) | RBS-pcyA | 796 | [13] |
| BBa_K1587003 | Basic part | crt | 786 | [1] [2] [3] [4] [7] |
| BBa_K1587004 | Basic part (others) | pBla-RBS-ccr-RBS-hbd-RBS-crt-RBS- tesB-RBS-atoB-Terminator | 5175 | [1] [2] [3] [4] [6] |
| BBa_K1587005 | Basic part (others) | pBla-RBS-hbd-RBS-crt-RBS- tesB-RBS-atoB-Terminator | 3827 | [1] [2] [3] [4] [6] |
| BBa_K1587006 | Basic part (others) | pOmpC-LacIbox-RBS-cI | 905 | [12][13] |
| BBa_K1587007 | Basic part (others) | RBS-pflB-RBS-pflA-Terminator | 3093 | [7] [8] [9] [10] [11] |
Attraction (butyrate pathway)
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.
Basic parts
tesB (BBa_K1587001)
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).
crt (BBa_K1587003)
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.
Other parts
ccr-Butyrate pathway (BBa_K1587004)
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.
Butyrate pathway wihout ccr (BBa_K1587005)
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).
Eradication (formate pathway)
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.
