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Applications of BBa_K542010

Datasheet for Part BBa_K542010 in E. coli strain BL21 (DE3).

Introduction

Lumazine synthase (LS) from Aquifex aeolicus forms icosahedral microcompartment (MC) assemblies of 60 or 180 monomeric units that self assemble and are capable of isolating proteins from their local environment. As shown in previously published work (1), the LS protein has been mutated so that the interior of the MC is negatively charged; the UL 2009 iGEM team has submitted the mutated LS gene to the parts registry (BBa_K249002). A negatively charged interior allows for preferential compartmentalization of positively charged molecules, which can easily be engineered through the addition of a poly-arginine tag to a target protein (1). The size of the cavity of the Lumazine Synthase microcompartment was enlarged and the negative charge was increased via directed evolution (2). The compartment formed by this polypeptide has a larger cavity and a larger net negative charge, thus allowing a larger loading capacity into the cavity of the compartment.

The part was synthesized by Bio Basic Inc. into the pET28a plasmid vector (which allows for expression of ELS with an N-terminal His-tag). The Enhanced Lumazine Synthase (ELS) was moved into the pSB1C3 vector by the Lethbridge 2011 team.

Characterization

Overexpression

The His-tagged ELS in pET28a was transformed into E. coli BL21 (DE3) and overexpressed.

Materials and Methods

4 X 500 mL of LB media in 2 L flasks were inoculated with E. coli BL21 (DE3) cells expressing ELS. Cells were grown at 37°C with shaking until the culture reached an OD₆₀₀ of 0.683. The cultures were then induced with 10 mM IPTG. Culture samples containing equal amounts of cells were taken before induction and 30 min, 1 hour, 2 hours, and 3 hours post induction for analysis by SDS-PAGE. At 5 hours post induction the cells were harvested by centrifugation at 5000 x g for 5 min. The cell lysate was resuspended in 42 mL of buffer containing 50 mM Tris pH 8.0, 60 mM NH₄CL, 7 mM β-mercaptoethanol, 1 mM PMSF, 7 mM MgCl₂, 300 mM KCl, 10 mM imidazole, and 15% glycerol. Afterwards, 0.05 g of crystallized lysozyme was added to the cell suspension and incubated on ice for 1 hour. The lysate was then centrifuged for 70 minutes at 30000 x g to obtain the S30 fraction.

Results

Figure 1. 12.5% SDS-PAGE of His-tagged ELS Overexpressed in E. coli BL21 (DE3). Samples were taken at 1 hour increments after IPTG induction. Lane 1: zero hours; Lane 2: 1 hour; Lane 3: 2 hours; Lane 4: 3 hours. The expected molecular weight of enhanced lumazine synthase is 18.5 kD.

Conclusion

Figure 1 clearly displays an increase in expression of a protein smaller than 25.0 kD, which is approximately the expected size of enhanced lumazine synthase monomers (18.5 kD).

Protein Purification

Enhanced lumazine synthase microcompartments were purified using size exclusion chromatography (SEC) to determine if the purified ELS formed a homogenous mixture of compartments, heterogenous mixture of microcompartments, or remained in monomer form.

Materials and Methods

In order to obtain a sample of ELS that was near homogeneity for characterization with SEC, the ELS sample was first purified by immobilized metal-ion affinity chromatography (IMAC). Ni²⁺-Sepharose beads supplied by Sigma Aldrich were pre-swelled in buffer containing 20% ethanol and 1mM NaCl. 3.75 mL of the Ni²⁺-Sepharose slurry was added to a 50 mL falcon tube. The beads were centrifuged at 500 x g for 2 min and the ethanol-containing buffer decanted. The beads were washed with 3 bed volumes of sterile water. The column was then washed in 6 bed volumes of Buffer A containing 50 mM Tris pH 8.0, 60 mM NH₄CL, 7 mM β-mercaptoethanol, 1 mM PMSF, 7 mM MgCl₂, 300 mM KCl, 10 mM imidazole, and 15% glycerol. S30 cell extract was applied to the column and mixed gently. The resin-S30 extract suspension was incubated on ice for 1 hour. The mixture was centrifuged at 500 x g for 2 min and the supernatant decanted. The supernatant was kept for further analysis by SDS-PAGE. The resin was washed 3 times with a full falcon tube volume (50 mL) of Buffer A. The mixture was centrifuged at 500 x g for 2 min and the supernatant decanted. The supernatant was kept for further analysis by SDS-PAGE. The column was washed 4 times with a full falcon tube volume (50 mL) of Buffer B (Buffer A plus 20 mM imidazole). The mixture was centrifuged at 500 x g for 2 minutes and the supernatant decanted. The supernatant was kept for further analysis by SDS-PAGE. The protein was eluted 9 times using 90% bed volume of Buffer E (Buffer A plus 250 mM imidazole). After each wash the sample was kept for analysis by SDS-PAGE.

Fractions 1 – 9 were pooled and concentrated using a Vivaspin Column with a 10 kDa molecular weight cut-off.

Concentrated protein samples from the Ni²⁺-Sepharose column were applied to a Sephacryl S400 size exclusion chromatography column at a flow rate of 0.4 mL/minute. 150 mL of SEC Buffer (50 mM sodium phosphate, 5 mM EDTA, 200 mM NaCl, pH 8.0, with 20% glycerol) was pumped through the column. The fractions eluted were collected and the absorbance was measured at 280 nm (see Fig 3 and 4).

Results

Figure 2. SDS-PAGE (15%) of ELS purification by IMAC. Lane 1: protein marker. Lanes 2-10: Elution Fractions 1 – 9.

Figure 3. (A) Concentrated protein sample from the Ni²⁺-Sepharose column applied to a Sephacryl S400 size exclusion chromatography column at a flow rate of 0.4 mL/minute. The absorbance of the eluting solution was measured at 280nm. (B) Magnified view of chromatogram of the two peaks seen in (A).

Conclusion

Figure 2 shows that with each wash proteins of a similar size were eluted off the resin. These proteins run at the expected molecular weight of enhanced lumazine synthase monomers (18.5 kD).

The chromatogram (Figure 3) from the chromatography experiment displays two regions of increased absorbance at 280 nm, between 82 and 135 mL of buffer eluted. These fractions were pooled, concentrated, and analyzed using transmission electron microscopy.

Transmission Electron Microscopy

Purified samples of enhanced lumazine synthase from size exclusion chromatography (SEC) were characterized using transmission electron microscopy. We viewed samples of each of the two peaks from the SEC; however only one sample contained microcompartments.

Materials and Methods

Purified Samples in solution were placed on a carbon grid and negatively stained using uranyl acetate. Carbon grids containing the samples were then viewed with a Hitachi H-7500 Transmission Electron Microscope. 

Results

Figure 4. Transmission electron microscopy of Enhanced Lumazine Synthase microcompartments.  Microcompartments of approximately 40 nm can be seen at x 20K magnification.

Figure 5. Transmission electron microscopy of Enhanced Lumazine Synthase microcompartments.  Microcompartments of approximately 40 nm can be seen at x 100K magnification.

Conclusion

As expected, TEM micrographs show polyhedral particles of approximately 40 nm, corresponding to the size described by Wörsdörfer et al. (2011). This confirms that E. coli is capable of producing enhanced lumazine synthase.

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) Viadiu H, Aggarwal AK (2000). Structure of BamHI bound to nonspecific DNA: a model for DNA sliding. Mol. Cell 5 (5): 889-895.
(2) Wörsdörfer, B., Woycechowsky, K.J., and Hilvert, D. (2011). Directed Evolution of a Protein Container. Science. 331: 589-592.

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