Part:BBa_K2213012

LacUV5_EutS_Tet_EutMN

There is an improved version of this part: https://parts.igem.org/Part:BBa_K3265026

LacUV5_EutS_Tet_EutMN is a composite of parts https://parts.igem.org/Part:BBa_K2213000 and https://parts.igem.org/Part:BBa_K2213001.

Figure 1: Schematic overview of LacUV5_EutS_Tet_EutMN (BBa_K2213012)

Characterisation

This part was co-transformed with EutLK-Low-PduD-mCherry-PPK (https://parts.igem.org/Part:BBa_K2213013) to form EutSMNLK+Low_PduD+PPK, and is characterized as follows.

A 24 hour induction was DAPI stained to determine microcompartment formation and tag localisation.


Figure 2: Fluorescence microscopy images of promoter-PduD associated mCherry and DAPI stained polyphosphate.



Figure 3. Visible light, mCherry and DAPI signals from DAPI stained E. coli, lacking PPK.

DAPI staining and mCherry fluorescence within E.coli appear to be colocalised, and locally enriched to foci at particular sub-cellular sites (Figure 2). This heterogeneous distribution of mCherry is induced by the presence of promoter-PduD-mCherry-PPK, with controls lacking the PPK element showing largely ubiquitous mCherry fluorescence throughout the cell (Figure 3). We conclude these results to indicate successful tag localisation and by proxy successful expression of Eut subunits.

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.

Our improved part with characterization can be found here: BBa_K3265026 (https://parts.igem.org/Part:BBa_K3265026)

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1260
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 4125
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3451