Difference between revisions of "Team:RDFZ-China/Safety"
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<p>Our project uses Bacillus subtilis 168. strain as the major chassis for the expression of sfp, yerP and LmrA. This strain is classified into Biosafety Level 1[1] based on the standards made by USA Center for Disease Control and Prevention. Therefore, we can use the chassis with relative ease, since the strain is non-pathogenic for healthy individuals and it poses minimal potential hazard to our lab members and the lab environment. But we still have to follow the biosafety precautions in our school lab.</p> | <p>Our project uses Bacillus subtilis 168. strain as the major chassis for the expression of sfp, yerP and LmrA. This strain is classified into Biosafety Level 1[1] based on the standards made by USA Center for Disease Control and Prevention. Therefore, we can use the chassis with relative ease, since the strain is non-pathogenic for healthy individuals and it poses minimal potential hazard to our lab members and the lab environment. But we still have to follow the biosafety precautions in our school lab.</p> | ||
| − | <h1> | + | <h1> Multidrug Resistance Factor LmrA </h1> |
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<p>LmrA is a ABC transporter responsible for multidrug resistance in Lactotococcus lactis. It can actively efflux a wide spectrum of clinically relevant antitumor, antimicrobial, antiviral drugs[2], including anthracyclines, vinca alkaloids, aminoglycosides, lincosamides, macrolides, quinolones, streptogramins, tetracyclines, chloramphenicol and novobiocin with energy provided by cellular ATP. Studies on heterologous expression of lmrA in Escherichia coli. has raised our awareness of potential biosafety issues. Transformation of the hypersensitive Escherichia coli strain CS1562 by a LmrA encoding plasmid has resulted in an increased resistance to 17 out of 21 clinically most used antibiotics[3], which is a significant result suggesting that such wild spectrum resistance is probable to be observed again in Bacillus subtilis, a Gram positive bacteria like Lactotococcus lactis, while Escherichia coli. is Gram negative. To reduce the risk as much as we can, we deliberately ordered LmrA coding sequence, without regulatory elements, to be synthesized by IDT and tried to assemble the operon afterward in one step to avoid unexpected expression of LmrA in E. coli. strain DH5a. Therefore, we have proposed a plan using a kill-switch regulated by the absence of crude-oil related hydrocarbons in our chassis so that the potential problem of unwanted resistance may be avoided if we use LmrA instead of endogenous YerP for surfactin efflux. </p> | <p>LmrA is a ABC transporter responsible for multidrug resistance in Lactotococcus lactis. It can actively efflux a wide spectrum of clinically relevant antitumor, antimicrobial, antiviral drugs[2], including anthracyclines, vinca alkaloids, aminoglycosides, lincosamides, macrolides, quinolones, streptogramins, tetracyclines, chloramphenicol and novobiocin with energy provided by cellular ATP. Studies on heterologous expression of lmrA in Escherichia coli. has raised our awareness of potential biosafety issues. Transformation of the hypersensitive Escherichia coli strain CS1562 by a LmrA encoding plasmid has resulted in an increased resistance to 17 out of 21 clinically most used antibiotics[3], which is a significant result suggesting that such wild spectrum resistance is probable to be observed again in Bacillus subtilis, a Gram positive bacteria like Lactotococcus lactis, while Escherichia coli. is Gram negative. To reduce the risk as much as we can, we deliberately ordered LmrA coding sequence, without regulatory elements, to be synthesized by IDT and tried to assemble the operon afterward in one step to avoid unexpected expression of LmrA in E. coli. strain DH5a. Therefore, we have proposed a plan using a kill-switch regulated by the absence of crude-oil related hydrocarbons in our chassis so that the potential problem of unwanted resistance may be avoided if we use LmrA instead of endogenous YerP for surfactin efflux. </p> | ||
Revision as of 03:17, 29 October 2017
About Our Chassis
Our project uses Bacillus subtilis 168. strain as the major chassis for the expression of sfp, yerP and LmrA. This strain is classified into Biosafety Level 1[1] based on the standards made by USA Center for Disease Control and Prevention. Therefore, we can use the chassis with relative ease, since the strain is non-pathogenic for healthy individuals and it poses minimal potential hazard to our lab members and the lab environment. But we still have to follow the biosafety precautions in our school lab.
Multidrug Resistance Factor LmrA
LmrA is a ABC transporter responsible for multidrug resistance in Lactotococcus lactis. It can actively efflux a wide spectrum of clinically relevant antitumor, antimicrobial, antiviral drugs[2], including anthracyclines, vinca alkaloids, aminoglycosides, lincosamides, macrolides, quinolones, streptogramins, tetracyclines, chloramphenicol and novobiocin with energy provided by cellular ATP. Studies on heterologous expression of lmrA in Escherichia coli. has raised our awareness of potential biosafety issues. Transformation of the hypersensitive Escherichia coli strain CS1562 by a LmrA encoding plasmid has resulted in an increased resistance to 17 out of 21 clinically most used antibiotics[3], which is a significant result suggesting that such wild spectrum resistance is probable to be observed again in Bacillus subtilis, a Gram positive bacteria like Lactotococcus lactis, while Escherichia coli. is Gram negative. To reduce the risk as much as we can, we deliberately ordered LmrA coding sequence, without regulatory elements, to be synthesized by IDT and tried to assemble the operon afterward in one step to avoid unexpected expression of LmrA in E. coli. strain DH5a. Therefore, we have proposed a plan using a kill-switch regulated by the absence of crude-oil related hydrocarbons in our chassis so that the potential problem of unwanted resistance may be avoided if we use LmrA instead of endogenous YerP for surfactin efflux.
