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Team:Glendale CC AZ/Project/Approach
From 2013.igem.org
PprI
First, 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). When we received all the biobricks, they were in the form of agar stabs. The cells from these stabs were inoculated into LB/chloramphenicol liquid media and incubated at 37ºC overnight. Next, the plasmid had to be isolated from the chassi, E. coli strain NBE 10β, so Minipreps were performed. The resulting products from the minipreps were run on a flash gel (link to data) verifying that we had the correct plasmid. After the verification step, growth curve experiments were designed 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 an IPTG-induced promoter, cultures containing no IPTG were used as control groups as well. In addition to growth curve experiments, survival plate experiments with the same conditions were also performed. However, these plate experiments were performed with a 10,000x dilution of the original bacterial culture to allow for quantifiable colony growth. Finally, we designed primers for use in PCR to amplify the PprI gene in D. hopiensis. Isolated D. hopiensis DNA was used with these primers and the resulting PCR reaction was run out on a Flash gel to confirm the amplification and presence of PprI in D. hopiensis (link to data).
LEA
Our team ordered the part BBa_K401000 submitted by the Valencia team. This part features the coding sequence of the PM2 gene which corresponds to the LEA3 proteins from soybean (Glycine max). We received E. coli strain NBE 10β transformed with the PM2 gene-containing plasmid in the form of an agar stab. We inoculated cells into LB/chloramphenicol liquid media and incubated them overnight at 37ºC. Next, we performed miniprep (link) and ran the products on a flash gel (link) which validated that we had the correct plasmid. We intended to include the PM2 biobrick in our desiccation toolkit but due to time we concentrated our work on Deinococcus.
D. radiodurans- Esther
D. hopiensis- Fern/Cristina
PprI- Andrew
PprA- Sean
PprM- Teresa
RecA- Teresa
Our Biobrick Construction
LacI/RBS/TT - Fern/Cristina
