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Team:Paris Bettencourt/Project/Target
From 2013.igem.org
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| - | + | <h2>Introduction</h2> | |
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We have designed a drug screen assay to specifically target the mycobacterial sulfite reductase protein SirA, using M.Smegmatis’ synthetic sulfite reduction pathway cloned into an E. coli chassi. SirA is essential for M. tuberculosis persistence phenotype as sulfur containing amino acids are particularly sensitive to oxidative stress within the macrophage and must regularly be replaced. In addition, a homolog within humans has not been found for SirA demonstrating why SirA has become a promising candidate as a drug target. Currently, there are no drug candidates that are known to specifically inhibit SirA. Conventional drug screens involve measuring growth inhibition of batch cultures of M. tuberculosis. However, this method does not provide information regarding the mechanism of drug action nor do compounds that inhibit exponential growth necessarily have an effect on persistent TB. Whilst a batch-culture model for persistence exists, there are numerous technical difficulties, such as maintaining the nutrient poor, microaerobic and oxidative conditions required for high-throughput applications. Our model overcomes the problem of the long doubling time of M. tuberculosis and informs us of specific inhibition of the sulfite reduction pathway. We plan to achieve this by comparing a drug screen of our E. coli construct against its parent E. coli prior to the deletion of the native sulfite reduction pathway. Any drug candidates that have activity against both the wild-type E. coli and our construct are non-specific inhibitors of E. coli growth. However, any drug candidates that inhibit only the growth of our E. coli construct will have activity specific for our cloned pathway. | We have designed a drug screen assay to specifically target the mycobacterial sulfite reductase protein SirA, using M.Smegmatis’ synthetic sulfite reduction pathway cloned into an E. coli chassi. SirA is essential for M. tuberculosis persistence phenotype as sulfur containing amino acids are particularly sensitive to oxidative stress within the macrophage and must regularly be replaced. In addition, a homolog within humans has not been found for SirA demonstrating why SirA has become a promising candidate as a drug target. Currently, there are no drug candidates that are known to specifically inhibit SirA. Conventional drug screens involve measuring growth inhibition of batch cultures of M. tuberculosis. However, this method does not provide information regarding the mechanism of drug action nor do compounds that inhibit exponential growth necessarily have an effect on persistent TB. Whilst a batch-culture model for persistence exists, there are numerous technical difficulties, such as maintaining the nutrient poor, microaerobic and oxidative conditions required for high-throughput applications. Our model overcomes the problem of the long doubling time of M. tuberculosis and informs us of specific inhibition of the sulfite reduction pathway. We plan to achieve this by comparing a drug screen of our E. coli construct against its parent E. coli prior to the deletion of the native sulfite reduction pathway. Any drug candidates that have activity against both the wild-type E. coli and our construct are non-specific inhibitors of E. coli growth. However, any drug candidates that inhibit only the growth of our E. coli construct will have activity specific for our cloned pathway. | ||
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| - | < | + | <h2>Flux Balance Analysis of Sulfite Reduction Pathway</h2> |
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We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth. Finally we introduced two new reactions for sirA and fprA and a new species fdxA. We found that growth was restored with the mycobacteria pathway resulting in a f value of 0.9105. | We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth. Finally we introduced two new reactions for sirA and fprA and a new species fdxA. We found that growth was restored with the mycobacteria pathway resulting in a f value of 0.9105. | ||
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| - | < | + | <h2>Synthetic Mycobacteria Pathway</h2> |
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We designed a synthetic M.smegmatis derived sulfite reduction pathway containing SirA; the sulfite reductase, and two supporting genes that are required for its function in E.coli; FdxA and FprA. FdxA is a mycobacterial Ferredoxin cofactor which is oxidised by SirA during the sulfite reduction reaction and FprA is a Ferredoxin-NADPH reductase use replenish the reduced Fdx pool. The sequence used as a template was taken from literature in which these genes were previously expressed in E. coli for purification and in vitro characterization of the enzyme kinetics with restriction sites removed and then codon optimized for expression in E. coli. These genes were then cloned into two Duet expression vectors, one containing sirA and one containing the supporting genes before being transformed into our knock out mutant strains of E. coli. | We designed a synthetic M.smegmatis derived sulfite reduction pathway containing SirA; the sulfite reductase, and two supporting genes that are required for its function in E.coli; FdxA and FprA. FdxA is a mycobacterial Ferredoxin cofactor which is oxidised by SirA during the sulfite reduction reaction and FprA is a Ferredoxin-NADPH reductase use replenish the reduced Fdx pool. The sequence used as a template was taken from literature in which these genes were previously expressed in E. coli for purification and in vitro characterization of the enzyme kinetics with restriction sites removed and then codon optimized for expression in E. coli. These genes were then cloned into two Duet expression vectors, one containing sirA and one containing the supporting genes before being transformed into our knock out mutant strains of E. coli. | ||
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| - | < | + | <img src="https://static.igem.org/mediawiki/2013/8/88/PB_final_Smegmatis.png" width="535px"/> |
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| + | <h2>Creation of Knock out Mutants</h2> | ||
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We prepared two strains of E. coli which have the sulfite reduction pathway deleted: BL21 (DE3) ΔCysI Δfpr ΔydbK and BL21 (AI) ΔCysI. CysI is responsible for sulfite reduction in E. coli, while fpr and ydbK are two non-essential genes that consume ferredoxin. These two genes are deleted, as sulfite reduction in mycobacteria is ferredoxin dependent in comparison to E. coli in which it is NADPH dependant. To ensure that these two genes do not interfere with our system, we deleted these genes as well. | We prepared two strains of E. coli which have the sulfite reduction pathway deleted: BL21 (DE3) ΔCysI Δfpr ΔydbK and BL21 (AI) ΔCysI. CysI is responsible for sulfite reduction in E. coli, while fpr and ydbK are two non-essential genes that consume ferredoxin. These two genes are deleted, as sulfite reduction in mycobacteria is ferredoxin dependent in comparison to E. coli in which it is NADPH dependant. To ensure that these two genes do not interfere with our system, we deleted these genes as well. | ||
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| + | <h2>Synthetic Corn Pathway</h2> | ||
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Additionally we prototyped the system with a reconstruction of a sulphite reduction pathway previously designed and published by the silver group (2011 Barstow et al). In place of CysI, a corn (Zea mays) derived sulfite reductase (zmSIR) was used. Two additional genes were included: Spinach ferredoxin (soFD), and corn derived ferredoxin NADP+ reductase (zmFNR). These genes, respectively, are required for production of the ferredoxin cofactor and the NADP+ ferredoxin reductase and are required for sulfite reductase (zmSIR) to function within E. coli. | Additionally we prototyped the system with a reconstruction of a sulphite reduction pathway previously designed and published by the silver group (2011 Barstow et al). In place of CysI, a corn (Zea mays) derived sulfite reductase (zmSIR) was used. Two additional genes were included: Spinach ferredoxin (soFD), and corn derived ferredoxin NADP+ reductase (zmFNR). These genes, respectively, are required for production of the ferredoxin cofactor and the NADP+ ferredoxin reductase and are required for sulfite reductase (zmSIR) to function within E. coli. | ||
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| + | <h2>Results</h2> | ||
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Upon successful cloning of the three genes into our E. coli deletion strain, we continued to confirm that all three genes are required for growth on minimal media. Our two synthetic pathways were found to rescue growth on a sulfurless amino acid supplemented minimal media. We hope that this technique of using synthetic biology to overcome problems faced in naturally occurring systems will be both a large boon to the pursuit of finding novel drug candidates in M. tuberculosis and more broadly as this technique can be used for high-throughput screening of any pathway that can be constructed to be essential for growth in E. coli. | Upon successful cloning of the three genes into our E. coli deletion strain, we continued to confirm that all three genes are required for growth on minimal media. Our two synthetic pathways were found to rescue growth on a sulfurless amino acid supplemented minimal media. We hope that this technique of using synthetic biology to overcome problems faced in naturally occurring systems will be both a large boon to the pursuit of finding novel drug candidates in M. tuberculosis and more broadly as this technique can be used for high-throughput screening of any pathway that can be constructed to be essential for growth in E. coli. | ||
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Revision as of 15:34, 4 October 2013
Background
Results
BioBricks
Aims
Introduction
We have designed a drug screen assay to specifically target the mycobacterial sulfite reductase protein SirA, using M.Smegmatis’ synthetic sulfite reduction pathway cloned into an E. coli chassi. SirA is essential for M. tuberculosis persistence phenotype as sulfur containing amino acids are particularly sensitive to oxidative stress within the macrophage and must regularly be replaced. In addition, a homolog within humans has not been found for SirA demonstrating why SirA has become a promising candidate as a drug target. Currently, there are no drug candidates that are known to specifically inhibit SirA. Conventional drug screens involve measuring growth inhibition of batch cultures of M. tuberculosis. However, this method does not provide information regarding the mechanism of drug action nor do compounds that inhibit exponential growth necessarily have an effect on persistent TB. Whilst a batch-culture model for persistence exists, there are numerous technical difficulties, such as maintaining the nutrient poor, microaerobic and oxidative conditions required for high-throughput applications. Our model overcomes the problem of the long doubling time of M. tuberculosis and informs us of specific inhibition of the sulfite reduction pathway. We plan to achieve this by comparing a drug screen of our E. coli construct against its parent E. coli prior to the deletion of the native sulfite reduction pathway. Any drug candidates that have activity against both the wild-type E. coli and our construct are non-specific inhibitors of E. coli growth. However, any drug candidates that inhibit only the growth of our E. coli construct will have activity specific for our cloned pathway.
Flux Balance Analysis of Sulfite Reduction Pathway
We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth. Finally we introduced two new reactions for sirA and fprA and a new species fdxA. We found that growth was restored with the mycobacteria pathway resulting in a f value of 0.9105.
Synthetic Mycobacteria Pathway
We designed a synthetic M.smegmatis derived sulfite reduction pathway containing SirA; the sulfite reductase, and two supporting genes that are required for its function in E.coli; FdxA and FprA. FdxA is a mycobacterial Ferredoxin cofactor which is oxidised by SirA during the sulfite reduction reaction and FprA is a Ferredoxin-NADPH reductase use replenish the reduced Fdx pool. The sequence used as a template was taken from literature in which these genes were previously expressed in E. coli for purification and in vitro characterization of the enzyme kinetics with restriction sites removed and then codon optimized for expression in E. coli. These genes were then cloned into two Duet expression vectors, one containing sirA and one containing the supporting genes before being transformed into our knock out mutant strains of E. coli.
Creation of Knock out Mutants
We prepared two strains of E. coli which have the sulfite reduction pathway deleted: BL21 (DE3) ΔCysI Δfpr ΔydbK and BL21 (AI) ΔCysI. CysI is responsible for sulfite reduction in E. coli, while fpr and ydbK are two non-essential genes that consume ferredoxin. These two genes are deleted, as sulfite reduction in mycobacteria is ferredoxin dependent in comparison to E. coli in which it is NADPH dependant. To ensure that these two genes do not interfere with our system, we deleted these genes as well.
Synthetic Corn Pathway
Additionally we prototyped the system with a reconstruction of a sulphite reduction pathway previously designed and published by the silver group (2011 Barstow et al). In place of CysI, a corn (Zea mays) derived sulfite reductase (zmSIR) was used. Two additional genes were included: Spinach ferredoxin (soFD), and corn derived ferredoxin NADP+ reductase (zmFNR). These genes, respectively, are required for production of the ferredoxin cofactor and the NADP+ ferredoxin reductase and are required for sulfite reductase (zmSIR) to function within E. coli.
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