Part:BBa_K769001
PompC-RBS-GFP-Double terminator
When OmpC promotor is activated by phosphorylated OmpR, GFP is expressed.
Usage and Biology
Characterization by team IGEM19_UI_Indonesia
This part contains the OMPC promoter and Green Fluorescent Protein at its downstream OmpC is the outer membrane porin that is only expressed when the environment osmolarity is high. Therefore, when the environment is in high osmolarity, OmpC promoter will be highly activated and express more GFP in the end. In this experiment, we would like to characterize 3 types of bacteria (TOP10, DH5a, and BL21) that contains this parts into various sodium concentration (Figure 1). The sodium concentration that we used were: 85.5 mM (low salt), 171mM (normal Luria-Bertani broth), 256.6 mM, 342 mM, and 427 mM.
Before performing this characterization, we had plotted the standard curve obtained from iGEM kit for fluorescence measurement. The results of this study were measured by using the same equipments and were performed under the same condition as when during the measurement using the standard curve. All of the results and values presented above were generated from Excel file provided by iGEM.
The plate reader results for the fluorescence intensity of GFP (MEFL/particles) in three bacterial strains TOP10, DH5a and BL21 are obtained from our plate reading after all of these bacteria were inoculated in LB broth for 12 hours (Figure 2). Essentially, all strains of bacteria showed increased fluorescence with increasing NaCl concentrations in LB medium with the highest expression at 427mM. Interestingly, all bacteria showed to have decreased fluorescence when sodium concentration were increased from 171 mM to 256.6 mM. We had searched multiple papers but questions remain unanswered. There need to be further studies to determine this phenomenon. As expected, when the sodium concentration was to be increased from 256.6 mM to 342 mM, fluorescence readings increased. Fluorescence readings were at its peak when sodium concentration were 427 mM.
TOP10 has relatively higher MEFL/particles to be compared with BL21. This is contradicting our thinking that TOP10 will have a lower expression compared to BL21 because BL21 is a protein-expressing-bacteria while TOP10 is bacteria mainly functional for increasing plasmid count (cloning). A possible reason for this phenomenon is that there are differences in ompC promoter regulation in different bacterial strains. According to a characterization done by SDU-Denmark, it is found that OmpR-regulated promoter showed a high leaky expression in high copy vectors (e.g. TOP10) compared to low copy vectors (e.g. BL21) confirming our findings in our characterization. From the results, DH5a then BL21 showed a gradual increase in expression with increasing NaCl concentrations in LB medium. This indicates that DH5a and BL21 has a much more efficient control of gene expression with the ompC promoter.
Through this results, we hope that other teams who are considering using this part will consider which bacteria that is most appropriate for their research.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 656
Illegal XhoI site found at 557 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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