Part:BBa_K5427012
pTac + RBS 1_12000
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_RBS1_sfGFP in pJUMP24
pTac_RBS1_SpiderSilk_sfGLP in pJUMP24
Characterization
pTac_RBS1_sfGFP in pJUMP24 Results
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.
pTac_RBS1_SpiderSilk_sfGLP in pJUMP24 Results
The results from our growth curve indicated that there was a significant difference between the two variables (empty vector vrs construct). Each culture was grown for 10 hours and Optical Density (OD) was taken every 2 hours at 600nm. The data showed that although both strains grew at a controlled rate, the empty vector seemed to outperform our construct. We saw that our construct variable’s exponential phase of growth lasted until around the 6 hour mark where we can see a beginning of plateauing into stationary phase. When we compared that with the empty vector growth, we saw a continuation of the stationary phase until the end of our experiment. Specifically at the 10 hour mark we observed a 48.03% reduction in growth of our vector in DH5a. We concluded that there must be some metabolic strain that our construct placed on the bacteria. Other literature has stated that in E.coli during silk protein synthesis upregulates stress response proteins, and therefore hinder growth significantly. Since we still observed growth, just significantly diminished, we determined that spider silk production would still be possible, but at a lower output than desired and that its growth rate does not affect biomass accumulation.
Figure 6 | Growth Curve for pTac_RBS 1_Spider silk_sfGFP_pJUMP 24 in E.coli strain DH5a and a DH5a empty vector control at 37 degrees grown in regular LB media. Each culture was grown for 10 hours and measurements of Optical Density (OD) at 600nm was taken every 2 hours. Each culture had 3 replicates grown and measured for OD, the average values were taken and plotted on the growth curve chart for analysis.
| None |