Part:BBa_K769001
PompC-RBS-GFP-Double terminator
When OmpC promotor is activated by phosphorylated OmpR, GFP is expressed.
Usage and Biology
Contribution: Universitas Indonesia 2019
Group: Universitas Indonesia / Authors: Raysha Farah, Eko Ngadiono
Highest expression of GFP in TOP10 E. coli strain due to differences in OmpC promoter regulation
Aims
The aim of this study is to characterize Part: BBa_K769001 which is a PompC-RBS-GFP-Double terminator in 3 different bacterial strains. In order to choose a suitable bacterial strain for our project, we used 3 different bacterial strains based on their cloning, subcloning and expression capabilities. For this study we will be using E. coli strains TOP10, which is a high efficiency strain ideal for cloning large plasmids and BACs so has a high copy vector, DH5a, which maximizes transformation efficiency too, and BL21, which is an ideal strain for expression vectors. The presence of ompC promoter in the BBa_K769001 construct may lead to different expression and regulation of GFP on these bacterial strains hence we use these variety of bacterial strains. We will be using a plate reader to measure the fluorescence of the transformed bacterial strains.
Experiments
This part contains the OmpC promoter, a promoter regulated by OmpR, and contains 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 characterized 3 types of bacteria (TOP10, DH5a, and BL21) that contains part (BBa_K769001) into various sodium concentration for 12 hours (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. We did not measure the OD600 of the bacteria because the value generated from iGEM fluorescence measurement kit is in MEFL/E.coli particles. Emission of GFP were read at 520nm. The experiment were performed using the same equipments and under the same condition as when during the measurement of the standard curve. All of the results and values presented above were generated from Excel file provided by iGEM. All step were followed according to iGEM's protocols.
Results
We confirmed the expression of GFP by using SDS PAGE in various bacteria and various osmolarity (Figure 2). The result of SDS-PAGE was consistent with our findings where the expression of GFP (26.9 kDA) was indeed increased from low osmolarity to high osmolarity (Figure 3).
Table 1. The Absorbance (OD 600) of TOP10, DH5a and BL21 bacteria transformed with OmpC/GFP (treated with various concentration of sodium) in duplicates
Table 2. The Fluorescence (520 nm) of TOP10, DH5a and BL21 bacteria transformed with OmpC/GFP (treated with various concentration of sodium) in duplicates
Table 3. The MEFL/ particles of TOP10, DH5a and BL21 bacteria transformed with OmpC/GFP (treated with various concentration of sodium) in duplicates
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 3). 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.
Discussion and Conclusion
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. OmpR-regulated promoter mediated gene expression even in the absence of its transcription factor.(1) According to a characterization done by SDU-Denmark (BBa_R0082), 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. (2) 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.
Reference:
1. Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, et al. Engineering Escherichia coli to see light. Nature [Internet]. 2005 Nov 23 [cited 2019 Oct 16];438(7067):441–2. Available from: http://www.nature.com/articles/nature04405
2. SDU Denmark. BBa_R0082. iGEM [Internet].https://parts.igem.org/Part:BBa_R0082#Contribution:_Hamburg_2016
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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