Part:BBa_K5427014
pTac + RBS 2_220
Background Information
RBS 1, 2, 3, 4 (alongside T7 inducible promoter) were previously tested for their efficiency to control and regulate synthetic operon containing bphS_bphO_yhjH genes (diguanylate cyclase (DGC), heme oxygenase (BphO) and phosphodiesterase (PDE), respectively.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Assembly Information
pTac_RBS2 in pJUMP24
pTac_RBS2_sfGFP in pJUMP24
Characterization
This experiment was used to compare the four different RBS’ by measuring both the growth of each strain of bacteria, and the amount of fluorescence generated by sfGFP in each sample. The best RBS would theoretically produce the strongest sfGFP signal/appearance and have minimal detrimental effects to the growth of the bacteria that it was transformed into. This experiment was repeated in two different growing conditions (30℃ and 37℃) to observe how temperature affected the plasmid containing bacteria. The conditions at 30℃ slowed the growth of each bacteria but it is unlikely that the transformed plasmids had any effect. There was no appreciable change in growth or generation of sfGFP for each RBS tested. While there was no statistical significance between the different RBS’ tested it seems that RBS 4 routinely resulted in less overall growth of each bacterial strain. On the opposite end of the spectrum RBS 1 seemed to have the most positive effect on bacterial growth overall
Figure 1 | Growth curve of pTac_RBS1/2/3/4_sfGFP_pJUMP24 vector in 4 different E.coli bacterial strains; A) DH5alpha, B) BL21, C) K12, and D) Rosetta Gami. These strains were cloned with our construct and measured for growth and 30℃ using optical density of 600 nm to measure the growth of each strain. OD measurements were taken every 2 hours for a total of 10 hours for each culture while growing in liquid LB.
Figure 2 | Growth curve of pTac_RBS1/2/3/4_sfGFP_pJUMP24 vector in 4 different E.coli bacterial strains; A) DH5alpha, B) BL21, C) K12, and D) Rosetta Gami. These strains were cloned with our construct and measured for growth and 37℃ using optical density of 600 nm to measure the growth of each strain. OD measurements were taken every 2 hours for a total of 10 hours for each culture while growing in liquid LB.
To determine overall production of sfGFP we performed a fluorometric characterization experiment. E.coli strain DH5alpha was induced with 3mM IPTG for 3 hours and subsequently lysed via French Press. Fluorescence was then measured for ribosome binding sites 1,2,3 and 4. Our results indicated that RBS1 showed a relatively strong fluorescence signal of 10.33, compared to RBS’s 2,3 and 4 signals of -0.66, -4.66, and -2.66, respectively. We then concluded that RBS1 produces sfGFP at a 1665.15% higher rate compared to RBS2. This result further confirms that RBS1 is the best ribosome binding site for our system when considering both growth and overall fluorescence production.
Figure 3 | Fluorescence (with blank subtracted) data for sfGFP isolated from DH5a containing the pTac_RBS 1/2/3/4_sfGFP_pJUMP 24 plasmid after 3 hour induction with 3 mM IPTG.
Figure 4 | Fluorescence (with blank subtracted) data for sfGFP isolated from DH5a containing the pTac_RBS 1/2/3/4_sfGFP_pJUMP 24 plasmid after overnight induction with 1 mM IPTG.
These pictures are all of the growth cultures of p(RBS 1,2,3,4_sfGFP_pJump24) in BL21, K12, DH5a, and R.Gammi. The fluorescence was measured after the 10 hour growth curve was performed. Notably, the only fluorescence was in two of the positive no-insert controls for the pJUMP24 plasmid, no other samples generated enough sfGFP to be detected by eye. The two vials containing the visible sfGFP left to right are DH5a and R.Gammi.
Figure 5 | Samples of RBS 1,2,3,4_sfGFP_pJump24 for BL21, K12, DH5a, and R.Gammi being tested for fluorescence.
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