In the list below you will find an overview over the BioBrick parts created by the iGEM Toulouse 2015 team and added to the IGEM registry.
For our project, we worked on three main biological modules : Attract, Eradicate, and Regulate to alternatively produce the two molecules of interest (butyrate by day, formate by night).
|Name||Type||Genic construction||Module||Length (bp)||Sequencing||References|
|BBa_K1587000||Composite (with RBS)||RBS-ho1||Regulate||744||Ok|| |
|BBa_K1587002||Composite (with RBS)||RBS-pcyA||Regulate||796||Ok|||
|BBa_K1587003||Basic part||crt||Attract||786||Sequenced until 736 pb : ok|||
|BBa_K1587009||Composite (with RBS)||POmpC-LacIbox-RBS-cI-RBS-pflB-RBS-pflA-Terminator||Eradicate||4006||-|||
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.
A gene from Escherichia coli (Accession Number: EG10995) 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).
A gene from Clostridium acetobutylicum (Accession Number: GJIH-2688) introduced in our bacterium after codon optimization in order to obtain a better expression in our strain. The crt enzyme substrate is 3-hydroxybutyryl CoA, and the product is Crotonyl CoA. This reaction does not need any coenzyme.
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 above. ccr encodes a crotonyl-CoA reductase (an oxidoreductase which acts on the double bond CH=CH). hbd from Clostridium acetobutylicum encodes a 3-hydroxybutyryl-CoA dehydrogenase (an oxidoreductase which catalyses the formation of alcohol function). atoB, from E. coli, encodes an acetyl-CoA acetyltransferase which catalyses the condensation of two acetyl-CoA.
Butyrate pathway wihout ccr (BBa_K1587005)
This BioBrick construction is the same as the previous one, but for the fact that it does not contain the ccr gene from Streptomyces collinus. It is composed of a constitutive promoter P(Bla) (BBa_I14018) and of 4 of the 5 genes present in the previous construction. Genes are issued from two micro-organisms : in yellow from E. coli, and in blue from Clostridium acetobutylicum. The green circles correspond to the strong RBS sequence (BBa_B0030) based on Ron Weiss thesis and the red one is the terminator (BBa_B1006).
Eradication (formate pathway)
Formate pathway (BBa_K1587007)
pflB encodes a 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
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 these two genes. For this, pflB and pflA are put together with two RBS (BBa_B0030) in front of them to allow the proteic synthesis. A strong terminator (BBa_B1006) ends up the sequence.
We aimed to produce butyric acid in our trap during
the day and formic acid during the night.
We designed a light response system which is improved to obtain an
on/off switch of genic expression.
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).
Ho1 with RBS (BBa_K1587000)
A 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 gene comes from the cyanobacterium Synechocystis sp. PCC 6803.
The biobrick is composed of a strong RBS (BBa_B0030) and Ho1 coding region (BBa_K566022).
PcyA with RBS (BBa_K1587002)
A gene required for chromophore synthesis in photosynthetic
light-harvesting complexes, photoreceptors, and circadian clocks. The gene comes from the cyanobacterium Synechocystis sp. PCC 6803.
The biobrick is composed of a strong RBS (BBa_B0030) and pcyA coding region (BBa_K566023).
Red light biosensor (BBa_K1587008)
Regulated formate production (BBa_K1587009)
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