Part:BBa_K3265026
EutSMN bacterial microcompartment
This is an improved version of part BBa_K2213012 from team Manchester 2017. We down-regulated the base expression by exchanging the RBS to a the very weak RBS J61100. Furthermore we replaced the LacI with the araBAD promoter system to get tighter control of the expression of the EutS protein, as the LacI is known to be leaky.
These changes were to enable production of all three Eut-proteins, EutS, EutM and EutN to enable the formation of bacterial micrompartments (BMC's) in E.coli. The problem with the original part was that the induced cultures grow much slower and showed a strong reduction in OD after 20 hours. This led to the hypothesis that the production of the EutN and EutM proteins are toxic to the bacteria, as bacteria only producing EutS have been shown to grow normally.
Here you can see the original measurements (Figure 1): https://parts.igem.org/Part:BBa_K2213000
Here you can see the part we improved: https://parts.igem.org/Part:BBa_K2213012
Usage and Biology
BMC's are currently being investigated and engineered to enable compartmentalization of biochemical reactions in bacteria: https://www.ncbi.nlm.nih.gov/pubmed/31235547
The first challenge is to produce functional BMC's with desired properties such as size, selective import/export of substrates and complexity. By first being able to produce the proteins that form these BMC's at desired levels without damaging the bacteria is an important step.
We could showcase that simply using a low binding affinity RBS such as J61100 is enough to regulate base expression of EutM and EutN proteins so that they are not toxic to the cell while using standard inducer concentrations.
Characterization
To test if our part could now produce the EutM and EutN proteins at levels low enough so that it would not be toxic to the cells anymore, we redid the OD measurement with the original EutSMN part.
Approach
First we streaked colonies of DH5alpha transformed with this part on Tetc and ATC plates to get an initial idea of how much we need to use for our liquid culture (initial screen)
After these results we went on to measure OD of inoculated liquid cultures over 20 hours.
Procedures
100mL LB cultures were inculcated with a single colony from an overnight transformation of pSEVA441 vector ( low copy number plasmid) carrying this part. E. coli strain DH5alpha was used.
Cultures were grown for 4 hours at 37° 300 rpm and then induced with 4.5 ng/mL Anhydrotetracylcine (ATC) or 45 ng/mL Tetracycline (Tetc) and 1mM Arabinose ((+)araB) each. One culture was not induced (control).
All concentrations are to be interpreted as final concentrations in the liquid culture. 45 ng/mL Tetc/ATC = 0.1 uM
Measurements were taken each hour for 9 hours and a final measurement was taken after 20 hours.
ATC was used at 10x less concentration since its a much stronger inducer than Tetc and has lower toxicity.
Results
Initial screen
We streaked DH5alpha transformed with our improved part on plates carrying high and low concentrations of Tetc and ATC to get an idea as to how much to use to induce our liquid cultures.
Spectinomycin was added due to the resistance gene in our backbone.
Surprisingly a concentration of 45 ng/mL Tetc (0.1 uM) seemed low enough that the cells actually survived. At 1 uM Tetc or ATC the bacteria didn't grow well at all. This supports the hypothesis that EutN and EutM are toxic for bacteria at high levels.
An ON liquid culture of a 0.1 uM Tetc colony was pelleted and showed fluorescence under UV light, a good indicator that the proteins were actually produced.
OD measurement
We did an OD measurement, using the information from the plate streak essay to use appropriate amounts of inducers.
It seems that our very weak RBS J61001 is doing a good job at keeping the base expression levels low enough so that not a lot of the protein is produced. OD increase is fairly similar to the control, even after induction and the OD decrease after 20 hours is not as strong as in the original part.
Imaging
We confirmed that the proteins were being produced by imaging with a confocal microscope, samples for imaging were taken 5 hours after induction.
induced:
uninduced:
Conclusion
Our improved part, when transformed in a low copy number plasmid, can be used to produce the proteins EutM and EutN at low enough concentrations to not cause strong stress on the bacteria but at high enough concentrations for form BMC's.
This information could be used to apply this approach to other Eut-protein parts such as https://parts.igem.org/Part:BBa_K2213002
For information about the vector and parts we used, please visit the "design" page.
Results
It seems that our very weak RBS J61001 is doing a good job at keeping the base expression levels low enough so that not a lot of the protein is produced.
Imaging
We confirmed with imaging that the protein is produced at high enough levels to form compartment:
This picture was taken 10 hours past induction, the signal does not seem very strong but there were a moderate number showing micrompartments.
The control was also imaged:
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1293
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1232
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1067
Illegal AgeI site found at 3824 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3212
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