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Team:Pitt/HSP60 Promoter/Methods
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<p>We constructed the Hsp60 promoter and Hsp60+TM4RBS parts by PCRing these two regions from a plasmid containing the promoter and TM4RBS (gift of G. Hatfull, University of Pittsburgh, Ref. 1) using complementary primers containing the biobrick suffix and prefix. The PCR products can be seen in Figure 1. below. We then digested the inserts using XbaI and PstI and ligated them into the biobrick submission vector pSB1C3. To perform a preliminary test of the promoter, we cloned the mRFP1 gene and the strong E coli RBS (B0034) downstream of the Hsp60 promoter to form Hsp60-RBS-mRFP1 (Part: BBa_K1548002). We then performed an induction experiment to determine whether the hsp60 promoter would be active in E. coli by growing Mach1 E. coli cells containing this plasmid in LB + Cam overnight. We then diluted the cultures 1:250 and grew them in triplicate for 4hrs at 37C. After this time we normalized the OD600 of the bacteria and read the mRFP1 fluorescence using a plate reader. From this experiment we observed no activity of the hsp60 promoter in <i>E. coli</i>. Following this result we performed an in silico <i>E. coli</i> promoter prediction analysis on the hsp60 promoter using the BPROM program in the Softberry software package (Link: <a href = "http://linux1.softberry.com/">http://linux1.softberry.com/</a>). This software predicted that the promoter would not function in <i>E. coli</i>, and thus our negative results in <i>E. coli</i> were validated.</p> | <p>We constructed the Hsp60 promoter and Hsp60+TM4RBS parts by PCRing these two regions from a plasmid containing the promoter and TM4RBS (gift of G. Hatfull, University of Pittsburgh, Ref. 1) using complementary primers containing the biobrick suffix and prefix. The PCR products can be seen in Figure 1. below. We then digested the inserts using XbaI and PstI and ligated them into the biobrick submission vector pSB1C3. To perform a preliminary test of the promoter, we cloned the mRFP1 gene and the strong E coli RBS (B0034) downstream of the Hsp60 promoter to form Hsp60-RBS-mRFP1 (Part: BBa_K1548002). We then performed an induction experiment to determine whether the hsp60 promoter would be active in E. coli by growing Mach1 E. coli cells containing this plasmid in LB + Cam overnight. We then diluted the cultures 1:250 and grew them in triplicate for 4hrs at 37C. After this time we normalized the OD600 of the bacteria and read the mRFP1 fluorescence using a plate reader. From this experiment we observed no activity of the hsp60 promoter in <i>E. coli</i>. Following this result we performed an in silico <i>E. coli</i> promoter prediction analysis on the hsp60 promoter using the BPROM program in the Softberry software package (Link: <a href = "http://linux1.softberry.com/">http://linux1.softberry.com/</a>). This software predicted that the promoter would not function in <i>E. coli</i>, and thus our negative results in <i>E. coli</i> were validated.</p> | ||
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Revision as of 18:57, 17 October 2014
Methods for HSP60
We constructed the Hsp60 promoter and Hsp60+TM4RBS parts by PCRing these two regions from a plasmid containing the promoter and TM4RBS (gift of G. Hatfull, University of Pittsburgh, Ref. 1) using complementary primers containing the biobrick suffix and prefix. The PCR products can be seen in Figure 1. below. We then digested the inserts using XbaI and PstI and ligated them into the biobrick submission vector pSB1C3. To perform a preliminary test of the promoter, we cloned the mRFP1 gene and the strong E coli RBS (B0034) downstream of the Hsp60 promoter to form Hsp60-RBS-mRFP1 (Part: BBa_K1548002). We then performed an induction experiment to determine whether the hsp60 promoter would be active in E. coli by growing Mach1 E. coli cells containing this plasmid in LB + Cam overnight. We then diluted the cultures 1:250 and grew them in triplicate for 4hrs at 37C. After this time we normalized the OD600 of the bacteria and read the mRFP1 fluorescence using a plate reader. From this experiment we observed no activity of the hsp60 promoter in E. coli. Following this result we performed an in silico E. coli promoter prediction analysis on the hsp60 promoter using the BPROM program in the Softberry software package (Link: http://linux1.softberry.com/). This software predicted that the promoter would not function in E. coli, and thus our negative results in E. coli were validated.
Figure 1. Gel image of PCR products for hsp60 promoter and hsp60+TM4 RBS.
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