Team:Stanford-Brown/SB16 BioMembrane Latex
Introduction
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Experimental design
In order to appropriate enhanced IPP and subsequently latex precursor production, we took advantage of Escherichia coli's endogenous MEP pathway, and implemented an HMG-CoA reductase pathway for enhanced IPP production. Both pathways result in the production of IPP and its isomer, DMAPP. However, the HMG-CoA pathway is exogenous to E. coli and was needed for enhanced production of IPP. While the MEP pathway converts pyruvate and G3P into IPP/DMAPP through a seven step process, the HMG-CoA (MVA) pathway converts acetyl CoA into IPP/DMAPP through a six step process. Both pathways employ the use of metabolic products produced and used by glycolysis and cellular respiration (calvin cycle); hence enhanced expression of these pathways is expected to affect cellular growth and development.
In the MEP pathway, the enzymes DXR and DXS are noted to be the rate limiting steps in the process, hence further enhanced production can be achieved via transfection of a high copy DXR/DXS gene. Since the rate limiting step in glycolysis is the conversion of oxaloacetate to phosphoenolpyruvate and CO2 by phosphoenolpyruvate carboxylkinase (PEPCK), including a vector with a high copy number for PEPCK could also enhance production of IPP through provision of more substrate.
Since the MEP pathway is already present in E. coli, the purpose of transfecting enzymes critical for the MEP pathway into E. coli is to increase the enzyme concentration in the cytosol, which should result in more enzymatic reactions taking place. For this purpose, the plasmid pMevT will be used in conjunction with pMevB to allow for production of Mevalonate from Acetyl-CoA, then IPP from Mevalonate. The following two plasmids are pSB1C3 plasmids with a CAT selection marker and T7 promoter. pMevT encodes the rate limiting enzymes for conversion of acetyl-CoA into Mevalonate, pMevB encodes the rate limiting enzymes for conversion of mevalonate to IPP. These have been adapted from Martin et al. [20]. pSB1C3 DXS consists of the DXS gene (DXP synthase) which is the rate limiting step in the endogenous MEP pathway.
For full realization of high throughput IPP production in E. coli, all three plasmids are to be transfected into the host to allow for high expression of all pathway elements. To verify all elements are property transfected, each plasmid must have a distinct selection markers such that with each subsequent transfection, un-transfected cells will be screened against. By transfecting one plasmid at a time into a population of cells with different selection assays, we can ensure that the final population of cells at the end of the process will contain all three genes.
Another item of concern is the metabolic strain each of these plasmids will place upon E. coli. IPP is a byproduct of glycolysis and cellular respiration; enhanced conversion of pyruvate and G3P or acetyl-CoA to IPP will decrease the amount of substrate available to the host for ATP production. Thus it is advisable to include a genetic switch, such as an IPTG inducible promoter into the plasmids. Other options include Pxtet tTA-On , lacR, lacO which can allow for regulated expression of downstream genes. For instance, lacR downstream of a Pxtet promoter on one construct and tTA-On downstream of a T7 promoter can allow for differential modulation of lacR downstream genes with Atc.
Using the same promoter sequence for each of the plasmids is also unadvisable, as identical promoters can result in homologous recombination (depending on host organism type), roadblocking, and RNAP leak through. The same issue applies to using the same terminators, in which RNAP leak through can occur and result in non-regulated expression of downstream genes. [33] Roadblocking is also an issue, in which a certain subset of repressors, when bound to a downstream promoter stopped by upstream transcription. This however can be easily addressed by moving regulatory elements upstream.
Since Martin et al. has already demonstrated use of the pMevB and pMevT gene in E. coli for the production of OPP and artemisinin, the only step we need to accomplish is transfection of a metabolic pathway and its enzymes that can convert IPP to cis 1,4 poly(isoprene). Prior literature suggests in the rubber tree Havea brasiliensis utilizes a rubber transferase for synthesis of latex polymers. [24] Additionally, H. brasiliensis rubber transferases require a supplement of glutathione, divalent magnesium ions, and SRPP proteins for production of rubber in detectable quantities. [29]
References
Since the MEP pathway is already present in E. coli, the purpose of transfecting enzymes critical for the MEP pathway into E. coli is to increase the enzyme concentration in the cytosol, which should result in more enzymatic reactions taking place. For this purpose, the plasmid pMevT will be used in conjunction with pMevB to allow for production of Mevalonate from Acetyl-CoA, then IPP from Mevalonate. The following two plasmids are pSB1C3 plasmids with a CAT selection marker and T7 promoter. pMevT encodes the rate limiting enzymes for conversion of acetyl-CoA into Mevalonate, pMevB encodes the rate limiting enzymes for conversion of mevalonate to IPP. These have been adapted from Martin et al. [20]. pSB1C3 DXS consists of the DXS gene (DXP synthase) which is the rate limiting step in the endogenous MEP pathway.
For full realization of high throughput IPP production in E. coli, all three plasmids are to be transfected into the host to allow for high expression of all pathway elements. To verify all elements are property transfected, each plasmid must have a distinct selection markers such that with each subsequent transfection, un-transfected cells will be screened against. By transfecting one plasmid at a time into a population of cells with different selection assays, we can ensure that the final population of cells at the end of the process will contain all three genes.
Another item of concern is the metabolic strain each of these plasmids will place upon E. coli. IPP is a byproduct of glycolysis and cellular respiration; enhanced conversion of pyruvate and G3P or acetyl-CoA to IPP will decrease the amount of substrate available to the host for ATP production. Thus it is advisable to include a genetic switch, such as an IPTG inducible promoter into the plasmids. Other options include Pxtet tTA-On , lacR, lacO which can allow for regulated expression of downstream genes. For instance, lacR downstream of a Pxtet promoter on one construct and tTA-On downstream of a T7 promoter can allow for differential modulation of lacR downstream genes with Atc.
Using the same promoter sequence for each of the plasmids is also unadvisable, as identical promoters can result in homologous recombination (depending on host organism type), roadblocking, and RNAP leak through. The same issue applies to using the same terminators, in which RNAP leak through can occur and result in non-regulated expression of downstream genes. [33] Roadblocking is also an issue, in which a certain subset of repressors, when bound to a downstream promoter stopped by upstream transcription. This however can be easily addressed by moving regulatory elements upstream.
Since Martin et al. has already demonstrated use of the pMevB and pMevT gene in E. coli for the production of OPP and artemisinin, the only step we need to accomplish is transfection of a metabolic pathway and its enzymes that can convert IPP to cis 1,4 poly(isoprene). Prior literature suggests in the rubber tree Havea brasiliensis utilizes a rubber transferase for synthesis of latex polymers. [24] Additionally, H. brasiliensis rubber transferases require a supplement of glutathione, divalent magnesium ions, and SRPP proteins for production of rubber in detectable quantities. [29]
References
