Part:BBa_K5427075
pTac+ RBS1_12000 + Spider Silk + sfGFP
Background Information
Our next step was to clone a Spider Silk producing gene into our previously tested plasmid (pTac_RBS1_sfGFP_pJUMP 24) whose growth, chassis, and vector have undergone optimization. To test the effect that this Spider Silk gene has in our construct we performed another growth curve at 37C, using DH5alpha as our bacterial strain.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1132
Illegal NheI site found at 1155 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1943
Illegal BsaI.rc site found at 1116
Illegal SapI.rc site found at 1211
Assembly Information
We tested the four previously constructed RBS-containing synthetic plasmid sequences, and the preliminary results indicated that RBS1 exhibited the highest growth potential for E. coli in LB media. Based on these findings, we selected RBS1 as the most suitable candidate for prokaryotic production of spider silk. Accordingly, we constructed the synthetic plasmid pJUMP24-pTac-RBS1-SpiderSilk-sfGFP using the following strategies. We linearized the vector pJUMP24-pTac-RBS1-sfGFP through PCR amplification to introduce Gibson assembly-compatible overlapping tail sequences, using forward and reverse primers #BBa_K5427034 and #BBa_K5427035, respectively. The forward primer was specifically designed to introduce a spacer between the sfGFP gene and the spider silk gene to provide the necessary structural flexibility. The spider silk gene block was ordered from IDT and amplified using primers #BBa_K5427036 (forward) and #BBa_K5427037 (reverse).
Characterization
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 1 | 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.
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