Difference between revisions of "Part:BBa K3265026"

Revision as of 17:35, 20 October 2019

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.

overview

We downregulated expression of the apparently toxic EutM and EutN proteins (by introducing a very weak RBS), as well as placing the EutS protein under a more tightly controlled promoter (araBAD). Our goal was to enable production of bacterial microcompartment proteins at levels that are not harmful to the cells. This is one option to enable formation of bacterial micro compartments (BMC's) so that they can be used for compound production in bacteria.

Approach

First, we redid the liquid culture OD measurement with the original EutSMN part to get a higher resolution compared to the original Manchester team data (check https://parts.igem.org/Part:BBa_K2213001 to see their data). Additionally we tested Anhydrotetracycline (ATC) as an inducer to rule out that the cells die because of Tetracycline(Tetc) which is potentially much more toxic to cells than ATC.

We then streaked colonies of DH5alpha transformed with our improved part on Tetc and ATC plates to get an initial idea of how much we need to use for the liquid culture of our improved part.

After these results we went on to measure OD of a inoculated liquid culture over 20 hours with our improved part.

Procedures

OD Measurement

100mL LB cultures were incoluated with a single colony from an overnight transformation of pSEVA441 vector ( low copy number plasmid) carrying the respective part. E. coli strain DH5alpha was used.

Cultures were grown for 4 hours at 37° 300 rpm and then induced with respective inducers and concentrations. One culture was always not induced (control). All concentrations are to be interpreted as final concentrations in the liquid culture.
For example: 45 ng/mL Tetc/ATC = 0.1 uM ATC

We used the same concentration for Tetc as the original Manchester measurement, however we also tried out ATC, to rule out a possible negative effect of Tetc on the cells since it can be toxic (while ATC has strongly reduced toxicity).
We used 10X less ATC since it is a stronger inducer than ATC. For (+) arabinose ( (+)araB) we also used the original concentration for all our measurements.

Results [original part]

Around 2 hours after induction the OD of the induced cultures increases slower compared to the control and the cultures reach a much lower OD maximum. This seems to fit the results of the Manchester measurement, except for the fact that their decrease in OD was much more drastic after 20 hours. We were not able to replicate that specific finding of theirs.

We therefore hypothesize that the production of the proteins EutM and EutN are detrimental to bacteria growth but don't necessarily kill them. EutN protein has been shown to be non toxic and not inhibit growth by Manchester team 2017.

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:

Results [improved part]

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 again 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.

These tests suggest that the production of the proteins EutN and EutM are detrimental to cell growth only at high levels and are NOT toxic.

With these results we have shown an improvement of the original part AND with the imaging we can also provide evidence that the part functions as it should.

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 so that BMC's can form.

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.

Sequence and Features