Team:Toulouse/Parts
Biobricks
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 Regulation to alternatively produce the two molecules of interest (butyrate by day, formate by 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 | Device | pBla-RBS-ccr-RBS-hbd-RBS-crt-RBS- tesB-RBS-atoB-Terminator | 5175 | [1] [2] [3] [4] [6] |
| BBa_K1587005 | Device | pBla-RBS-hbd-RBS-crt-RBS- tesB-RBS-atoB-Terminator | 3827 | [1] [2] [3] [4] [6] |
| BBa_K1587006 | Device | POmpC-LacIbox-RBS-cI | 905 | [12][13] |
| BBa_K1587007 | Device | RBS-pflB-RBS-pflA-Terminator | 3093 | [7] [8] [9] [10] [11] |
| BBa_K1587008 | Device | cph8 | 2288 | REF A METTRE !!! |
| BBa_K1587009 | Composite (with RBS) | POmpC-LacIbox-RBS-cI-RBS-pflB-RBS-pflA-Terminator | 4006 | [7] [8] [9] [10] [11] [12] [13] |
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)
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).
crt (BBa_K1587003)
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.
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.
Formate pathway (BBa_K1587007)
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.
Circadian swich
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).
Figure: Phycobilin biosynthetic pathway in cyanobacteria showing the formation of PCB from heme. The first step comprises Ho1- catalyzed heme degradation (Okada et al 2009).
Ho1 with RBS (BBa_K1587000)
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).
PcyA with RBS (BBa_K1587002)
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).
POmpC-LacIbox-RBS-cI (BBa_K1587006)
This biobrick contains OmpC promoter (BBa_R0082), a lacI box separated from cI gene by a RBS sequence (BBa_B0030).
Red light biosensor (BBa_K1587008)
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
Regulated formate production (BBa_K1587009)
Uses a positively regulated promoter (PompC) 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).
References
- [1] Layton DS & Trinh CT (2014) Engineering modular ester fermentative pathways in Escherichia coli. Metabolic Engineering 26: 77–88
- [2] Saini M, Hong Chen M, Chiang C-J & Chao Y-P (2015) Potential production platform of n-butanol in Escherichia coli. Metabolic Engineering 27: 76–82
- [3] Volker AR, Gogerty DS, Bartholomay C, Hennen-Bierwagen T, Zhu H & Bobik TA (2014) Fermentative production of short-chain fatty acids in Escherichia coli. Microbiology (Reading, Engl.) 160: 1513–1522
- [4] Saini M, Wang ZW, Chiang C-J & Chao Y-P (2014) Metabolic Engineering of Escherichia coli for Production of Butyric Acid. J. Agric. Food Chem. 62: 4342–4348
- [5] Hunt MC & Alexson SEH (2002) The role Acyl-CoA thioesterases play in mediating intracellular lipid metabolism. Prog. Lipid Res. 41: 99–130
- [6] Aboulnaga E-H, Pinkenburg O, Schiffels J, El-Refai A, Buckel W & Selmer T (2013) Effect of an oxygen-tolerant bifurcating butyryl coenzyme A dehydrogenase/electron-transferring flavoprotein complex from Clostridium difficile on butyrate production in Escherichia coli. J. Bacteriol. 195: 3704–3713
- [7] Thesis: SUNYA Sirichai. July 2012. Dynamique de la réponse physiologique d’Escherichia coli à des perturbations maîtrisées de son environnement : vers le développement de nouveaux outils de changement d’échelle. Ingénieries Enzymatique et Microbienne.
- [8] CEA-CNRS- Aix Marseille Université. February 2015. Paris . Activation d’enzymes bactériennes pour convertir le CO2 en source d’énergie renouvelable.
- [9] Crain AV & Broderick JB (2014) Pyruvate formate-lyase and its activation by pyruvate formate-lyase activating enzyme. J. Biol. Chem. 289: 5723–5729
- [10] Wei X-X, Zheng W-T, Hou X, Liang J, Li Z-J, Wei X-X, Zheng W-T, Hou X, Liang J & Li Z-J (2015) Metabolic Engineering of Escherichia coli for Poly(3-hydroxybutyrate) Production under Microaerobic Condition. BioMed Research International, BioMed Research International 2015, 2015: e789315
- [11] Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, Davidson EA, Scouras A, Ellington AD, Marcotte EM & Voigt CA (2005) Synthetic biology: Engineering Escherichia coli to see light. Nature 438: 441–442
- [12] Okada K (2009) HO1 and PcyA proteins involved in phycobilin biosynthesis form a 1:2 complex with ferredoxin-1 required for photosynthesis. FEBS Lett. 583: 1251–1256
- [13] Lee JM, Lee J, Kim T & Lee SK (2013) Switchable gene expression in Escherichia coli using a miniaturized photobioreactor. PLoS ONE 8: e52382
