Part:BBa_K542008:Experience

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Applications of BBa_K542008

Datasheet for Part:BBa_K542008 in E. coli strain DH5alpha.

Introduction

The intended purpose of the Lumazine Synthase microcompartment device is to specifically target proteins that have been tagged with a positively charged oligopeptide into the cavity formed by the microcompartment. Furthermore, multiple unique proteins may be targeted into the cavity (1) for the purposes of increasing the efficiency of a metabolic pathway, to name just one example. This test construct is designed to demonstrate that proteins with positively charged tags can be localized into the cavity of the microcompartment formed by the oligomerization of Lumazine Synthase monomers.

The expression of Lumazine Synthase and the fluorescent proteins are independently regulated by two separate promoters. Lumazine Synthase is regulated by the pLacI promoter and the fluorescent proteins are regulated by the pBAD inverter. Since the two are independently controlled, Lumazine Synthase microcompartments may be formed in the presence or absence of the fluorescent proteins (ie. absence or presence of arabinose, respectively).

Because the fluorescent proteins are tagged with a positively-charged poly-arginine tag and the Lumazine Synthase is mutated with a negative interior (BBa_K249002 and Lethbridge 2009 Modeling), enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) should be targeted into the microcompartments. ECFP and EYFP are a well known FRET pair.

Construct

Part Number BBa_K542008

The construct was made by assembling BBa_K542004 with BBa_K542005.

Figure 1. BBa_K542008. This construct contains pLacI Regulated Lumazine Synthase and pBAD Inverse-Regulated Arg-tagged ECFP and EYFP.

Characterization

Electron Microscopy

E. coli DH5α cells expressing BBa_K542008 and controls were viewed using Transmission Electron Microscopy (TEM).

Materials and Methods

E. coli DH5α cells containing BBa_K542008 as well as control cultures containing only plasmid pSB1C3 were grown overnight at 37°C. Approximately 400 mL of these cells were then resuspended in 5 mL SOC media, and induced with 5 mL or 10 mL of 1 M IPTG. Cells were again incubated at 37°C for approximately 4 hours. Cells were then spun down at 6000 x g for 2 min.

Cells were resuspended in 500 mL gluteraldehyde/formaldehyde fixative and incubated at room temperature for 2 hours. The solution was then spun at 6000 x g for 2 min.

The cell pellet was resuspended in 0.1 M sodium cacodylate buffer and incubated for 10 min. The solution was centrifuged for 2 min at 6000 x g. This step was repeated an additional 2 times. Cells were then resuspended in 1% osmium tetroxide fixative in 0.1 M CaC buffer and incubated at room temperature for 1 hour. The solution was centrifuged at 6000 x g for 2 min. Cells were then resuspended in MilliQ H2O.

The cells were spun down at 10000 x g for 5 min, and resuspended in 4% BIO-RAD standard low melting temperature agarose at 60°C. A drop of this cell-agarose mixture was then added to slides in order to harden, and incubated in MilliQ H₂O overnight.

Agarose-cell mixtures were cut into cubes of approximately 1 mm x 1 mm x 1 mm. They were then incubated in increasing alcohol concentrations up to 100% anhydrous ethyl alcohol. The cubes were then incubated in decreasing concentrations of alcohol and increasing concentrations of resin, up to 100% resin. The cubes were left in 100% resin to harden overnight.
The resin-embedded cubes of cells were thin-sectioned and placed on a carbon grid. They were stained with uranyl acetate and then positively stained with lead citrate and viewed under TEM.  

Results

Figure 2. E. coli DH5α control cells (left) and cells expressing BBa_K542008 (right) as viewed under transmission electron microscope.

Figure 3. E. coli DH5α cells expressing BBa_K542008 as viewed under transmission electron microscope.

Discussion and Conclusion

We found that while the controls had a fairly uniform interior, the lumazine expressing cells did not (Figure 2). In the lumazine cells there were homogenous areas of grey as well as darker spots, whereas the controls contained a more uniform interior.

We hypothesized that the lumazine accumulated together, which caused the isolation of other cellular elements. If this is the case, the microcompartments should most likely be the homogenous areas of grey not seen in the controls, and can’t be distinguished by contrast staining because they are aggregated together.

To see if this is the case we will look at cells expressing BBa_K542008 under the spectral confocal (fluorescence) microscope. We hypothesize that if the microcompartments are segregated from other cellular elements then the fluorescent proteins will also be segregated and therefore we should see areas within the cell with brighter fluorescence.

Fluorescence Microscopy

E. coli DH5α cells containing BBa_K542008 were viewed with fluorescence microscopy. This was done in order to determine if the distribution of fluorescent proteins within the cells was similar to the homogenous grey regions seen in the cells under TEM.

Materials and Methods

E. coli DH5α cells containing the BBa_K542008 construct were grown overnight at 37°C. They were spun down at 10,000 rpm and then resuspended in 1X phosphate buffer saline (PBS). The solution was centrifuged at 10,000 rpm, decanted and resuspended in 4% paraformaldehyde (PFA) overnight. This solution was then centrifuged at 10000 rpm and cells were resuspended in glycerol. The glycerol-cell mixtures were then placed on slides, coverslipped and sealed.

The slides were viewed with an Olympus FV1000 spectral confocal at 60X magnification. Pictures were taken using the ECFP and EYFP filters.

Results

Figure 4. E. Coli DH5a cells containing the BBa_K542008 construct as viewed under spectral confocal microscope with 60X magnification. The left picture corresponds to the ECFP filter and the right picture corresponds to the EYFP filter.

Conclusion

Figure 4 shows that the fluorescent proteins were expressed in the cells. It is also evident that the fluorescence was segmented in a way that looks similar to that found in the homogenous grey areas in the TEM micrographs of the cells.

References

(1) Seebeck, F., Woycechowsky, K., Zhuang, W., Rabe, J., and Hilvert, D. (2006). A simple tagging system for protein encapsulation. Journal of the American Chemical Society. 128: 4516-4517.

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