Team:Glendale CC AZ/Wetlab/Overview/Experiments

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==PprI==
==PprI==
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In 2011, Osaka team reported success increasing ionizing radiation resistance  in ''E. coli'' by transforming the bacteria with PprI, the transcriptional regulator involved in the expression of many DNA damage response proteins in ''Deinococcus radiodurans''. In order to determine if PprI would also increase desiccation resistance in ''E. coli'', our team decided to perform a series of experiments . To begin the project, the PprI expression device was ordered from the IGEM parts registry. This biobrick, identified as part BBa_K602005, features LacI constitutive  promoter (R0010), transcriptional regulator PprI (K602000) and ribosome binding site (B0034).  In addition to the PprI expression device, our team had other candidates biobricks for our experiments that were ordered at the same time. We received all the biobricks in the form of agar stabs that were inoculated into LB/chloramphenicol liquid media. The liquid cultures were incubated overnight at 37ºC. The next step was to isolate the plasmid from the chassi, E. coli strain NBE 10β; thus, miniprep[[https://2013.igem.org/Team:Glendale_CC_AZ/Protocols]] was performed. The products were run on a flash gel (link to data) verifying that we had the correct plasmid. After the verification step, we designed growth curves experiments using sodium chloride (NaCl) as the DNA damaging agent (link to data). For the NaCl growth experiments (link to protocols), we used liquid cultures that were inoculated with cells from the agar stabs received from IGEM. The experimental group was ''E. coli'' transformed with BBa_K602005 while the control group was  the same strain of ''E. coli'' but transformed with BBa_K602003. Registry part BBa_K602003 was used as the control group because it features only the coding sequence of RecA, a component of the DNA double-strand break repair mechanism in ''Deinococcus radiodurans''. Since the plasmid had IPTG-induced promoter, cultures containing no IPTG were used as control groups as well.
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In 2012, Osaka team investigated increasing Hydrogen Peroxide resistance  in ''E. coli'' by transforming the bacteria with PprI, the transcriptional regulator involved in the expression of many DNA damage response proteins in ''Deinococcus radiodurans''. In order to determine if PprI would also increase desiccation resistance in ''E. coli'', our team decided to perform a series of experiments. To begin the project, the PprI expression device was ordered from the IGEM parts registry. This biobrick, identified as part BBa_K602005, features LacI constitutive  promoter (R0010), transcriptional regulator PprI (K602000) and ribosome binding site (B0034).  In addition to the PprI expression device, our team had other candidates biobricks for our experiments that were ordered at the same time. We received all the biobricks in the form of agar stabs that were inoculated into LB/chloramphenicol liquid media. The liquid cultures were incubated overnight at 37ºC. The next step was to isolate the plasmid from the chassi, E. coli strain NBE 10β thus [https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/AlkalineLysis miniprep] was performed. The products were run on a flash gel (link to data) verifying that we had the correct plasmid. After the verification step, we designed [https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/GrowthCurve growth curve experiments] using sodium chloride (NaCl) as the DNA damaging agent (link to data). For these growth experiments, we used liquid cultures that were inoculated with cells from the agar stabs received from IGEM. The experimental group was ''E. coli'' transformed with BBa_K602005 while the control group was  the same strain of ''E. coli'' but transformed with BBa_K602003. Registry part BBa_K602003 was used as the control group because it features only the coding sequence of RecA, a component of the DNA double-strand break repair mechanism in ''Deinococcus radiodurans''. Since the plasmid had IPTG-induced promoter, cultures containing no IPTG were used as control groups as well. In addition to growth curve experiments, we also ran [https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/SurivalGrowth survival plate experiments] with the same conditions and it produced similar results.

Latest revision as of 17:16, 6 August 2013


Experiments

PprI

In 2012, Osaka team investigated increasing Hydrogen Peroxide resistance in E. coli by transforming the bacteria with PprI, the transcriptional regulator involved in the expression of many DNA damage response proteins in Deinococcus radiodurans. In order to determine if PprI would also increase desiccation resistance in E. coli, our team decided to perform a series of experiments. To begin the project, the PprI expression device was ordered from the IGEM parts registry. This biobrick, identified as part BBa_K602005, features LacI constitutive promoter (R0010), transcriptional regulator PprI (K602000) and ribosome binding site (B0034). In addition to the PprI expression device, our team had other candidates biobricks for our experiments that were ordered at the same time. We received all the biobricks in the form of agar stabs that were inoculated into LB/chloramphenicol liquid media. The liquid cultures were incubated overnight at 37ºC. The next step was to isolate the plasmid from the chassi, E. coli strain NBE 10β thus miniprep was performed. The products were run on a flash gel (link to data) verifying that we had the correct plasmid. After the verification step, we designed growth curve experiments using sodium chloride (NaCl) as the DNA damaging agent (link to data). For these growth experiments, we used liquid cultures that were inoculated with cells from the agar stabs received from IGEM. The experimental group was E. coli transformed with BBa_K602005 while the control group was the same strain of E. coli but transformed with BBa_K602003. Registry part BBa_K602003 was used as the control group because it features only the coding sequence of RecA, a component of the DNA double-strand break repair mechanism in Deinococcus radiodurans. Since the plasmid had IPTG-induced promoter, cultures containing no IPTG were used as control groups as well. In addition to growth curve experiments, we also ran survival plate experiments with the same conditions and it produced similar results.

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