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.

Fluorescent Resonance Energy Transfer (FRET)

Materials and Methods

Preparation of Cell Cultures and Controls
A total of six 5ml cell cultures of Escherichia coli DH5α cells containing the BBa_K542008 construct were grown under different conditions overnight at 37°C. As controls parts BBa_K542006, BBa_K331031, BBa_K331033, BBa_K542001 were grown normally overnight at 37°C.

Table 1. E.coli DH5α cells cultures grown in LB media and incubated overnight with shaking at 37°C. Samples #1-6 was grown up differently with specific treatments for the expression of certain parts within the BBa_K542008 construct. Samples #7-10 was grown as controls.

The following morning, six 50mL cultures were inoculated with 1mL of overnight culture grown to sample 6 (with nothing expressed), and four 50mL cultures were inoculated with samples 7-10. The 50mL cultures were allowed to grow with shaking at 37oC until they reached an OD600 of 0.6 (approximately 3 hours). Samples 1-3 and 6-10 were immediately pelleted by centrifugation (4oC, 5000xg, 20min) and placed on ice. Samples 4 and 5 were treated as described in table above. Pellets were weighed, and resuspended in 10mL/mg wet weight of the following buffer:

• 50mM TrisHCl (pH8.0)
• 250mM NaCl
• 5mM 2ME

In order to evaluate expression levels, 1mL of cells was removed from each culture, and analysed using SDS-PAGE (12%).

Lysis of Cells and Clearing of Cell Lysate
Resuspended cell cultures were incubated on ice, and subjected to 3x20 pulse sonication bursts, with 3 minute rests on ice.
Lysate was cleared by centrifugation (4C, 30000xg, 1 hour). Cleared cell lysate was removed and retained for fluorescent analysis.

Scanning of Cell Lysate using a Spectrofluorometer
Using a spectrofluorometer, 3 mL of cell lysate was used for each scan. Each scan was done at 5nm excitation and 5nm emission slits. For the analysis of the expression of Förster resonance energy transfer (FRET) within the micro compartment, each cell lysate was excited at 439nm and the emission spectra was read from 444nm to 650nm.

Results

Expression levels of Lumazine Synthase and fluorescent proteins

Load order for the 12% denaturing polyacrylamide gel is as follows:

1. Low Range Molecular Marker
2. K542008 – No Expression (Sample 6)
3. K542001 – Lumazine Synthase Control (Sample 8)
4. K331031 – YFP Control (Sample 9)
5. K331033 – CFP Control (Sample 10)
6. K542006 – Fluorescent Proteins (Both YFP and CFP) Control (Sample 7)
7. K542008 – Expression of fluorescent proteins only (Sample 1)
8. K542008 – Expression of Lumazine Synthase only (Sample 2)
9. K542008 – Co-expression of Lumazine Synthase and fluorescent proteins (Sample 3)
10. K542008 – Fluorescent proteins first, then Lumazine Synthase (Sample 4)
11. K542008 – Lumazine Synthase first, then fluorescent proteins (Sample 5)

Figure 1. Expression patterns of K542008 and controls

In every K542008 sample, regardless of treatment, lumazine synthase (16.6kDa) is expressed. This means that we are unable to control production of lumazine synthase as we had hoped. By altering the promoter, we hope to be able to control the production of lumazine synthase in order to evaluate how the expression patterns of lumazine synthase and arginine tagged proteins affect efficiency of co-localization of tagged proteins within the compartment.

The expression of the fluorescent proteins (each approximately 27kDa) can easily been seen in lanes 2 and 3. Although it is difficult to see fluorescent proteins in K542008 samples, fluorescence observed in FRET experiments suggests that protein is expressed.

FRET

By using sample 7 (fluorescent protein control; BBa_K542006) as the baseline for expression of fluorescent proteins in the cleared cell lysates, we evaluated samples 1-5 for FRET by comparing fluorescence at 475nm (CFP) and 528nm (YFP).

Figure 2. Relative change in fluorescence at 475nm and 528nm, when excited by light at 439nm. "LS then FP" is Sample 5; "FP Only" is Sample 1; "LS Only" is Sample 2; "FP then LS" is Sample 3; "LS then FP" is Sample 4.

In each sample, we observed a decrease in fluorescence at 475nm and a simultaneous increase in fluorescence at 528nm. We believe that this decrease in CFP fluorescence (observed at 475nm) and concomitant increase in YFP fluorescence (observed at 528nm) is indicative of FRET interactions occurring between CFP and YFP. The presence of FRET interactions naturally leads us to the conclusion that the oligo-arginine-tagged CFP and YFP are being co-localized within the lumazine synthase microcompartment.
Within the FRET results, the condition where lumazine synthase was expressed before the fluorescent proteins were expressed shows lower levels of fluorescent change than the other conditions, suggesting that fluorescent proteins have difficulty entering the completely formed microcompartments.

Conclusion

1. Proteins that have an oligo-arginine tail fused to their C-termini are localized within the microcompartment formed by the oligomerization of lumazine synthase proteins
2. Proteins can localize within the compartment more easily when they are co-expressed with lumazine synthase

Proteins have a more difficult time entering the compartment when the compartment is fully formed

Discussion

While we hoped to be able to control the expression of the fluorescent proteins and lumazine synthase temporally, our SDS-PAGE gel indicates that even in the absence of IPTG, the lac-inducible lumazine synthase is being expressed. This means we are not able to achieve one of our conditions – fluorescent proteins expressed first, then lumazine synthase. The constant expression of lumazine synthase gave us several measurements with co-expression of lumazine synthase and fluorescent proteins. Furthermore, co-expression approximates the condition we were attempting for our first condition – namely, the formation of microcompartments around tagged proteins. We were also able to measure the condition where lumazine synthase was expressed in the absence of fluorescent proteins, which were expressed after the production of the lumazine synthase microcompartments. We interpret the results to indicating that tagged proteins have more difficulty entering pre-formed lumazine synthase microcompartments.

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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