Part:BBa_K2213012

LacUV5_EutS_Tet_EutMN
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)

Results

According to our experiments, we were successful in achieving a significantly lower base level expression of the EutN and EutM proteins leading to a strong increase in liquid culture growth and higher OD 20 hours after induction compared to the original part.

First, we better did an liquid culture OD measurement with the original EutSMN part to get a higher resolution compared to the original Manchester team data.

Procedure

OD Measurement

100mL LB cultures were incoluated with a single colony from an overnight transformation of pSEVA441 vector carrying our improved EutSMN 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

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 fits to 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 this finding.

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:

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