Part:BBa_K5427070
J23119 + RBS 1_12000 + KerDZ
Background Information
Once our construct was created with our keratin degradation enzyme, we transformed this plasmid into DH5alpha, BL21, and Rosetta Gami, and performed a growth curve analysis. Salt stress may have a small effect on the cell metabolism, therefore we grew each strain in regular and low salt LB. Cultures were grown for 10 hours and Optical Density (OD) was taken every 2 hours at 600nm.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Assembly Information
We constructed a synthetic plasmid to express the keratin hydrolase, KerDZ. The KerDZ gene, originally derived from Actinomadura viridilutea strain DZ50, was synthesized and ordered from IDT. The pJUMP28-J23119-RBS1 vector backbone underwent sequential double digestion using the restriction enzymes SpeI and PstI. To introduce compatible restriction sites for pJUMP28, the KerDZ gene block was amplified via PCR using the forward primer P_pre10 (iGEM 2023 Designation #BBa_K4755025) and the reverse primer P_suf10 (iGEM 2023 Designation #BBa_K4755027). Both primers were sourced from the UAlberta iGEM 2023 project’s primer collection. Successful amplification was confirmed by agarose gel electrophoresis. The PCR product was subsequently double-digested with XbaI and PstI and ligated into the vector using NEB T4 DNA Ligase according to standard protocols.
Characterization
The growth curves of each E.coli strain in regular LB and low salt LB categorized the metabolic effect of salt between each strain, as well as highlighting the overall growth rate. Figure 1 showed the effects of each strain of E.coli when growing in regular LB which allowed us to examine the effect our plasmid had on each strain. We then concluded that our KerDZ construct grows most effectively in DH5alpha and BL21, notably at 10 hours we saw a 51.85% and 23.67% increase in growth, respectively, in comparison to Rosetta Gami at the same time mark. Although we observed adequate growth from both BL21 and DH5alpha, when compared to the empty vector controls DH5alpha under performed, whereas BL21 grew more than its control. These results are consistent with what we observed previously where BL21 grew better than its control and DH5alpha the opposite. Therefore we determined that either BL21 or DH5alpha would be suited for transformation of our vector. We did not exhibit any significantly large inhibitions of growth between any strain. We then performed this experiment again with cells incubating in Lennox LB (low sodium). Figure 2 indicates the same growth factors as seen in figure 1. Even in low salt concentrations KerDZ grew the most in DH5alpha, and then BL21, where Rosetta Gami performed the worst. Consistent with our previous growth curve, BL21 grew better than its empty vector control, and DH5alpha grew less than its control. These tests confirm previous results and allow us to conclude that either BL21 or DH5alpha would give us adequate growth with our constructs. We then compared figure 1 and figure 2 to determine if there was any significant difference in salt concentrations on growth. Comparing the OD measurements at 10 hours for each strain between low salt conditions and high, we determined that these cells grew better in regular LB. Rosetta Gami at 10 hours of growth had an average OD measurement of 0.38 in regular LB but a measurement of 0.269 in Lennox LB, meaning we saw a 29% increase in growth for regular LB. Similarly, BL21 showed a 16.6% increase in growth in regular LB at the 10th hour. Contradictorily, Rosetta Gami when comparing both regular and Lennox LB saw a 0.25% reduction in growth in the regular LB at hour 10. These results indicate that there is very little if any salt stress happening in the cells for regular LB compared to Lennox. We then compared this result to previous phases, figure 2 growth curve which tests a similar construct but without KerDZ, where we saw an increase in growth during the low salt condition. Since in figure 2 we saw the opposite results, where lower salt inhibited the growth of our organism, we determined that the KerDZ construct must have a higher tolerance to salt concentrations within the media, which allowed the plasmids to grow better in higher salt than seen in previous experiments. We can also conclude that there is very little effect that salt concentration has on the overall growth of the E.coli strains, and there was no real significant difference between the variables themselves.
Figure 1 | Growth Curve for J23119_RBS 1_KerDZ_pJUMP 28 in DH5a, R. Gami and BL21 at 37 degrees in Regular LB. Cultures were grown for 10 hours and Optical Density (OD) was taken every 2 hours at 600nm.
Figure 2 | Growth Curve for J23119_RBS 1_KerDZ_pJUMP 28 in DH5a, R. Gami and BL21 at 37 degrees in Lennox LB Cultures were grown for 10 hours and Optical Density (OD) was taken every 2 hours at 600nm.
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