Part:BBa_M50003
StickyGreen
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StickyGreen (BBa_M50003) is a bicistronic construct allowing linked expression of eCPX-nSA-cNano (BBa_M50000, a cell surface display protein containing two streptavidin binding domains, and mNeonGreen (BBa_M50002), a bright green fluorescent protein. A spacer separates the two genes (BBa_M50001). Both genes are preceded by a strong RBS (BBa_B0034). We hypothesized that this design would allow linked expression of these two proteins, resulting in bacterial strain that could both bind to streptavidin magnetic beads and fluoresce green (hence the name Sticky Green). However, this construct unfortunately does not function as designed.
Contents
Introduction
In recent years, new bioremediation strategies have been created, many of which rely on releasing genetically engineering bacteria into the contamination site to carry out the detoxification. For example, metabolic engineering has enabled the creation of hydrocarbon degrading bacteria, which can be deployed to clean up oil spills[1]. However, with such strategies comes the risk of inadvertly damaging the ecosystem due to the newly released organism. Thus, it is relevant to examine new ways of selectively retrieving these strains from the environment once bioremediation is complete[2][3].
One such strategy involves engineering bacteria to bind to magnetic beads, thus allowing easy retrieval of the desired strain using a magnet. In order for effective capture of the bacteria, a strong ligand/substrate system must be used; one commonly used system is the streptavidin binding peptide (SBP)/streptavidin pair[4]. By expressing a streptavidin binding peptide on the cell surface, bacteria should bind to commercially available streptavidin coated magnetic beads. Once the beads are collected by a magnet, the bound bacteria can be eluted off using biotin (which outcompetes the SBP for binding sites on streptavidin) and can be disposed of properly.
Part Design
Current bacterial display systems comprise of two parts: a scaffold protein that is anchored to the outer membrane, and a passenger peptide to be displayed[5]. Here, we chose to display streptavidin-binding domains on the outer membrane of E. coli using Rice and Daugherty's engineered membrane protein eCPX as scaffold. Both N and C terminus of eCPX are displayed on the extracellular side of the outer membrane, allowing for the possibility of biterminal fusion with streptavidin-binding domains, allowing for stronger ligand-substrate interactions and therefore more effective bacterial capture[6]. Because an N-terminal fusion of eCPX with a 15 residue streptavidin-binding domain had already been created (eCPX-nSA), the streptavidin-binding peptide NanoTag was fused to the remaining free C-terminus of eCPX-nSA to form eCPX-nSA-cNano. See BBa_M50000 for sequence) [4]. In order to monitor expression of eCPX-nSA-cNano, the bright green fluorescent protein (GFP) mNeonGreen (see BBa_M50002 is placed downstream of eCPX-nSA-cNano to form a bicistronic construct, henceforth termed StickyGreen[7}. Thus, expression of eCPX-nSA-cNano is linked with expression of mNeonGreen. Translation of mNeonGreen is driven by the strong ribosome binding site BBa_B0034. Expression of the two-gene transcript will be under the control of an IPTG inducible T5 lac promoter. In order to prevent steric effects and ribosome collisions, we will place a fifteen base pair spacer sequence BBa_M50001 between eCPX-nSA-cNano and mNeonGreen. In total, our novel gene StickyGreen spans 1432 base pairs (Figure 1). In order to express StickyGreen, the gene was cloned into the plasmid pD441-SR (DNA2.0), which encodes the kanamycin resistance selectable marker as well as the IPTG inducible promoter.
Methods
Creation of strains
StickyGreen was constructed via de novo synthesis and cloned into the vector pD441-SR by DNA2.0. We will henceforth refer to this plasmid pStickyGreen. The lyophilized plasmid was resuspended in 20 µL deionized water, and then 2.5 µL of this solution was transformed into chemically competent E. coli BW25113 (See Supplementary Information for protocol). Transformants were selected for kanamycin resistance by plating on Luria Broth (LB) supplemented with 50 µg/mL kanamycin (kan) plates at 37˚C. This new strain will herein be referred to as BW25113:pSG. In addition to transforming pStickyGreen into BW25113, we also transformed pSC101-rrbp1-mCherry (a plasmid constitutively expressing mCherry) and p15A-pBBa_J23100-eCPX-SBP (a plasmid constitutively expressing eCPX with an N-terminal streptavidin-binding domain) using the same KCM transformation protocol. These transformants were selected by plating on LB plates supplemented with 20 µg/mL chloramphenicol (cm), as these two plasmids conferred resistance to this particular antibiotic. The new strain containing pSC101-rrbp1-mCherry will herein be referred to as BW25113:RFP, and the strain containing p15A-pBBa_J23100-eCPX-SBP will be referred to as BW25113:control.
Characterization of inducible promoter
In order to characterize the IPTG-inducible promoter driving StickyGreen expression, a dose-response assay was performed. In a 96-well culture plate, IPTG at varying concentration from 0 µM to 1000 µM (1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.9, 0 µM) was added to 1mL LB/kan, then seeded with approximately 1x107 cells containing pStickyGreen. A total of six replicates of this assay was set up on the plate, then the plate was incubated at 37˚C in a shaker incubator for ~16 hours. Each sample was then diluted 1:10 in LB/kan and transferred onto a new 96 well plate for OD600 and GFP fluorescence measurements with a plate reader.
Pull-down assay
In order to characterize StickyGreen's potential for bacterial capture, a pull-down assay was performed. Overnight cultures of E. coli strains containing pStickyGreen (induced with 1mM IPTG final concentration, overnight), pSC101-rrbp1-mCherry, and p15A-pBBa_J23100-eCPX-SBP were inoculated from glycerol stocks into LB media containing the appropriate antibiotic, and grown for ~16 hours at 37˚C in a shaker-incubator. Once grown up, the absorbance at 600 nm was measured, then 1x109 cells of each strain were spun down at 13000g for 1 minute and each resuspended in 1mL of 1xPBST buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4, 0.05% Tween 20) to make working stocks of cells. From the working stocks, the mixtures detailed in Table 1 were made.
| Mixture | BW25113:pSG (µL) | BW25113:control (µL) | BW25113:RFP (µL) | Beads (µL) |
|---|---|---|---|---|
| Control 50:50 | 0 | 50 | 50 | 40 |
| Control 90:10 | 0 | 10 | 90 | 40 |
| pStickyGreen 50:50 | 50 | 0 | 50 | 0 |
| pStickyGreen 90:10 | 10 | 0 | 90 | 40 |
| RFP 100% | 0 | 0 | 100 | 40 |
Once the beads were added, the mixtures were placed on a rocker at room temperature for 15 minutes to allow cells to bind to the beads. Small samples of the cells and beads were taken to image at this point. The tubes were then placed on a magnetic separation rack (New England Biolab 6-Tube Magnetic Separation Rack, catalog number S1506S) and allowed to sit for 5 minutes at room temperature for beads to separate. The supernatant was then removed and imaged. Next, the beads were washed three times by adding 500 µL of 1x PBST, placing the tubes on a rocker for 5 minutes then separating the beads and removing the supernatant. After the washes were complete, 500 µL of biotin solution (1x PBST + 2mM biotin) was added and the tubes placed on the rocker for 15 minutes at room temperature to elute the cells off the beads. The beads were pulled down once more using the magnetic rack, and the supernatant transferred to clean tubes. The supernatant was then spun down at 13000g for 5 minutes, and the pellet resuspended in 20 µL of 1xPBST in order to concentrate cells. This solution was then imaged. In order to perform the pull down assay on exponential phased cells, the overnight cultures were diluted 1:100 in 5mL LB supplemented with the appropriate antibiotic, and grown at 37˚C with shaking for 2-3 hours (until OD600 = 0.4-0.6). BW25113:pSG was then induced with IPTG to a final concentration of 1mM, and allowed to grow for an addition hour. The cells were then spun down and resuspended in 1x PBST to a OD600 = 1 to make working stocks of cells. The pull down assay then proceeded identically to that performed on the stationary phase cells.
Microscopy
| Channel | Exposure (ms) |
|---|---|
| Phase | 20 |
| Cherry | 100 |
| FTIC | 150 |
Samples for microscopy were prepared by placing 2µL of liquid sample onto a 3mmx3mm agar pad, and allowed to dry for 10 minutes at room temperature. Agar pads were prepared by melting 0.15g agarose into 10 mL 1x phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4), then 700µL of the agarose solution gently poured onto a 22mmx22mm glass coverslip. Then, another 22mmx22mm glass coverslip was carefully placed on top to form an coverslip-agar-coverslip "sandwich". Once solidified, the coverslips were removed, and an approximately 3mmx3mm square was cut with a razor blade from the large agar pad (Figure 2).
Once dried, the agar pad containing the sample to be imaged was placed sample-side down onto coverglass chamber (Lab-Tek 1 Chambered Borosilicate Coverglass System). The sample was then imaged on a Nikon Ti-E inverted epifluorescence microscope, using a 60x oil-immersion objective with 1.5x magnifier. Images were taken in three channels: phase contrast, Cherry, and FTIC. Exposure times are listed in Table 2. Multiple fields of view from each sample were taken.
Cell lysate pull-down and SDS-PAGE
In order to test the localization of eCPX-nSA-cNano, we lysed induced BW25113:pSG cells as well as BW25113 and uninduced BW25113:pSG cells, then performed affinity chromatography to capture any protein that bound by streptavidin. SDS-PAGE was used to analyze the results.
To produce cell lysate, overnight cultures of BW25113 and BW25113:pSG were diluted 1:100 in 100 mL LB supplemented with the appropriate antibiotic (two cultures of BW25113:pSG were set up, for a total of three 100 mL cultures). The cultures were incubated at 37˚C and shaken at 180 rpm for 3 hours. After 3 hours, one of the BW25113:pSG cultures was induced by adding IPTG to a final concentration of 1mM. All three cultures were allowed to grow for another 3 hours. The cultures were then spun down at 5000 rpm for 5 minutes at 4˚C, and the supernatant discarded. The pellet was then resuspended in 4mL lysozyme buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 10 mg/mL lysozyme). The mixture was incubated at 37˚C for 30 minutes, then sonicated at 50% amplitude in three 30 second intervals, with 10 second rest periods between the pulses. To remove cell debris and insoluble proteins, the lysates were spun down at 4700 rpm for 5 minutes at 4˚C. The supernatant was then transferred in to a clean 15mL conical tube, and phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 1mM. A 1 mL sample of each lysate was set aside for SDS-PAGE. The remaining lysate samples were used for affinity chromatography with streptavidin magnetic beads. For each 3mL of lysate, 100 µL of beads were added. The solutions were placed on a rocker at 4˚C for 30 minutes for proteins to bind to the beads. The beads were then spun down, and the supernatant removed. The beads were washed three times with 500µL of wash buffer (1x PBST containing 1mM PMSF) by rocking the mixture for 15 minutes at 4˚C, spinning down the beads, then removing the supernatant. Protein was eluted off the beads with 100µL of 1x PBST containing 2mM biotin.
SDS-PAGE was then performed on these samples. A 4-12% gradient gel (BioRad MiniProtean TGX Gel, 10 wells, 1mm) was run at 200V for 40 min. In preparation for staining, gel was placed in deionized water and microwaved for 30 seconds. Gel was then placed in SimplyBlue SafeStain, microwaved for 30 seconds, and shaken for 5 minutes. Destaining occurred overnight by placing the gel in deionized water.
Material sources
pStickyGreen was synthesized via a combination of de novo synthesis and cloning by the company DNA 2.0. The plasmids pSC101-rrbp1-mCherry and p15A-pBBa_J23100-eCPX-SBP were obtained from the Covert lab. The host strain BW25113 was also provided by Covert lab. Streptavidin beads were purchased from New England Biolabs (catalog number S1420S). Biotin powder was obtained from Sigma-Aldrich. All microscopy material (coverslips, coverglass chamber, etc...) were obtained in the Covert lab. All other buffers and solutions were obtained from or made in the Uytengsu Teaching Lab.
Results
Characterization of inducible promoter
We measured the GFP fluorescence and OD600 for each of the samples, generating the plot shown in Figure 3. The GFP fluorescence and OD600 were normalized to the LB control, and then GFP fluorescence per well was normalized to OD600 to account for differences in cell density. Figure 3 indicates a dose-dependent response with apparent full induction of construct expression at 1mM IPTG.
Pull-down assay
Given that clear GFP expression was seen in the IPTG induction assay, we then proceeded to test whether we could pull down cells expressing StickyGreen using streptavidin magnetic beads, and verify the functionality of our novel eCPX-nSA-cNano protein design. In order to test the capacity of BW25113:pSG to be purified from a mixture of cells, we mixed this strain with a strain constitutively expressing mCherry (BW25113:RFP) in varying ratios. As a positive control, we also attempted to pull down cells constitutively expressing eCPX-nSA from a similar mixture. Finally, as a negative control, we also performed the pull down assay on BW25113:RFP to determine the amount of cells obtained during pull down due to non-specific binding. This experiment was carried out first with stationary phase cells, then with exponential phase cells to identify if growth phase affected pull down efficiency.
In order to quantify pull down efficiency, we counted the number of cells for each cell population within a 1000x1000 pixel field of view for each of the experimental groups, before and after pull down. A Chi-Square test of independence was then performed to determine if the cell population composition changed significantly after pull down. As expected, little to no cells were isolated from the negative control, indicating the the streptavidin beads display little to no non-specific binding. In both positive control groups (Control 50:50 and 90:10), the tagged cell populations were significantly enriched (see Table 3 and Table 4), validating that bacterial capture using the unmodified eCPX-nSA is both possible and effective. However with BW25113:pSG, little to no cells were pulled down with the streptavidin beads, indicating that the addition of the C-terminal Nano Tag has disrupted functionality of eCPX-nSA in some way. Growth phase does not seem to affect the efficiency of pull down, and pStickyGreen fails to grant E. coli the ability to bind to streptavidin magnetic beads.
| Red cells: Before | Tagged cells: Before | Red cells: After | Tagged cells: After | p-value | |
|---|---|---|---|---|---|
| Control 50:50 | 68 | 59 | 1 | 131 | <0.001* |
| Control 90:10 | 140 | 16 | 3 | 84 | <0.001* |
| pStickyGreen 50:50 | 100 | 67 | 1 | 1 | 0.778 |
| pStickyGreen 90:10 | 70 | 2 | 3 | 1 | 0.026 |
| Red cells: Before | Tagged cells: Before | Red cells: After | Tagged cells: After | p-value | |
|---|---|---|---|---|---|
| Control 50:50 | 76 | 61 | 1 | 523 | <0.001* |
| Control 90:10 | 152 | 24 | 7 | 357 | <0.001* |
| pStickyGreen 50:50 | 115 | 69 | 1 | 0 | 0.439 |
| pStickyGreen 90:10 | 96 | 1 | 8 | 1 | 0.398 |
SDS-PAGE
The failure to pull down BW25113:pSG could be attributed to several possibilities. Because fluorescence under the GFP channel was observed by confocal microscopy of induced BW25113:pSG cells, we know that the StickyGreen transcript is being expressed. The problem must therefore lie post-transcriptionally, leaving the following possibilities: 1) eCPX-nSA-cNano localized to the outer membrane, but the SA-binding domains lost function 2) eCPX-nSA-cNano failed to localize to the outer membrane but the SA-binding domains remain functional 3) eCPX-nSA-cNano failed to localize to the outer membrane and the SA-binding domains lost function. In order to determine which of the above scenarios was the cause of failure, we performed a pull down assay on cell lysate and visualized the eluted proteins on SDS-PAGE.
From the SDS-PAGE gel (Figure 6), it appears that no detectable amount of protein was isolated using the streptavidin beads (note that the faint bands in the lanes corresponding to the pull down samples are likely due to spill over of the whole cell lysates from the neighboring lanes). While this should rule out the case of a misfolded signaling domain that prevents localization but preserves function, we also expect bands corresponding to the size of eCPX-nSA-cNano (~24.1 kDa) as well as mNeonGreen (~26.6 kDa) in the induced BW25113:pSG cell lysate lane. However, we observed no apparent difference between the protein bands of the pre and post induction BW25113:pSG lysates, nor do we see a difference between the BW25113:pSG lysates and the lysate of the host strain BW25113. It is possible that after 3 hours post induction, mNeonGreen and eCPX-nSA-cNano have not reach sufficient concentrations within the cells to be detected in the whole cell lysate, or that the corresponding bands are simply washed out by the abundance of other proteins in the whole cell lysate. Other issues may include failure to induce expression, as well as overexpression resulting in aggregation into insoluble inclusion bodies that would be excluded from visualization in this procedure. A Western blot would be necessary to correctly identify the bands corresponding to mNeonGreen and eCPX-nSA-cNano.
In a second attempt to determine the localization of eCPX-nSA-cNano, we lysed cells and solubilized the cell debris pellet to sample the membrane proteins, then visualized the fractions on SDS-PAGE. Unfortunately, we again observed little difference in the pattern of protein bands in the lysates (Figure 7). The results are therefore inconclusive, and the cause of pull down failure remains unknown.
Discussion
In this project, we created pStickyGreen, a plasmid expressing a bicistronic transcript encoding mNeonGreen and eCPX-nSA-cNano. We hypothesized that this design would allow linked expression of these two proteins, resulting in bacterial strain that could both bind to streptavidin magnetic beads and fluoresce green (hence the name Sticky Green).
To begin, our transformation of pStickyGreen into \textit{E. coli} strain BW25113 was successful, and we yielded colonies that were kanamycin resistant. From the IPTG dose-response assay, we observed increasing green fluorescence with increasing IPTG concentration, consistent with the phenotype expected of the StickyGreen gene.
Although we did observe green fluorescence post-induction, we did not succeed in pulling down cells nor did we significantly enrich BW25113:pSG cells (p > 0.001). Because we observed correct behavior in our controls (successful pull down with BW25113:control, and little to no cells captured with BW25113:RFP), we can be sure that BW25113:pSG does not exhibit binding affinity for streptavidin.
Due to the bicistronic design of StickyGreen, the transcriptional expression of mNeonGreen is forcibly linked with that of eCPX-nSA-cNano; because we observe clear GFP signal in BW25113:pSG under confocal microscopy post-induction, the eCPX-nSA-cNano transcript must also be expressed. The problem therefore lies post-transcriptionally.
While our whole-cell pull down for BW25113:pSG failed, we were able to successfully pull down our positive control strain expressing eCPX with only an N-terminal streptavidin binding domain. This suggests that C-terminal fusion of the 15 residue NanoTag disrupts the protein structure of eCPX-nSA resulting in loss of function.
Disruption to the secondary and/or tertiary structures of eCPX-nSA due to the addition of the NanoTag may have caused loss of function by either failing to translocate to the outer membrane or causing loss of streptavidin affinity in the binding domains (or both). Because we were unable to isolate any detectable amount of protein in our cell lysate pull down experiment, we can conclude that no protein with affinity for streptavidin is expressed significantly in induced BW25113:pSG.
In order to obtain more information on the localization of eCPX-nSA-cNano, we ran another SDS-PAGE gel on both the cell lysates and the resulting cellular debris pellet (resolubilized using saturated urea solution). However, due to the abundance of other proteins in the samples, we could not draw conclusions on the localization of eCPX-nSA-cNano.
On the whole, we face difficulty in fully characterizing the StickyGreen system. We have yet to fully determine the nature of our limited cell capture success, but our functional positive control with N-terminal modification and the expression of GFP suggest that the eCPX scaffold is less robust for biterminal fusion than reported[6]. A more thorough characterization of final conformation may be enlightening in explaining how a C-terminal fusion may contribute to protein misfolding or loss of function.
In conclusion, our construct was unable to perform as intended. In the future, there are many experiments that can be done in order to both improve the efficacy of our construct and troubleshoot to pinpoint our current problems. First, in order to troubleshoot our protein, it is possible to tag our construct with a fluorescent tag for better visualization. This would allow us to determine if our construct is being translated, independent of the expression of GFP. Next, because GFP could be accumulating in the cell regardless of how our construct is being expressed/degraded, we can add a degredation tag to GFP so that it will closely match the levels of construct in the cell. Moreover, since there could have been problems due to inconsistent ribosome binding sites, it would be beneficial to change the pStickyGreen code so that the RBSs for the bicistron match. Either this would solve the expression problem, or it would rule out translation, allowing us to pinpoint post-translational folding as the problem.
References
[1] Das, N., Chandran, P. 2011. Microbial Degradation of Petroleum Hydrocarbon Contaminants: An Overview. Biotechnology Research International.
[2] Mayat, Z.; Hassanshahian, M.; Cappella, S. 2015. Immobilization of microdes for bioremediation of crude oil polluted environments: a mini review. Open Microbiol. J. 9:48-54.
[3] Ron., E. Z.; Rosenberg, E. 2014. Enhanced bioremediation of oil spills in the sea. Curr. Opin. Biotechnol. 27:191-194.
[4] Lamla, T.; Erdmann, V. A. 2004. The Nano-Tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins. Protein Expression and Purification. 33:39-47.
[5] van Bloois, E.; Winter, R.T.; Kolmar, H.; Fraaije, M.W. 2011. Decorating microbes: surface display of proteins on Escherichia coli. Trends in Biotechnology. 29(2):79-86.
[6] Rice, J. J.; Daugherty, P. S. 2008. Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides. Protein Eng. Des. Sel. 21(7):435-442.
[7] Shaner, N. C.; Lambert, G. G.; Chammas, A.; Ni, Y.; Cranfill, P. J.; Baird, M. A.; Sell, B. R.; Allen, J. R.; Day, R. N.; Israelsson, M.; Davidson, M. W.; Wang, J. 2013. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nature Methods. 10:407-409.
Supplementary Information
KCM Competent Cell Production
Grow cells overnight and dilute 1:100 in the morning in 1mL LB. Grow cells to OD600 = 0.5 (~2-3 hours). Cool cells down on ice to for 20-30 minutes, then centrifuge at 5000 rpm for 5 minutes. Resuspend cells in TSS buffer (20.4 mL LB, 1.25 mL DMSO, 167 µL 3M MgCl2, 3.2 mL 67% w/w PEG-6000 solution). Aliquot 50µL cells in to individual tubes. Use immediately or freeze at -80˚C.
KCM Transformation Protocol
Thaw and aliquot 50µL KCM competent cells on ice. Add 12.5µL of 5x KCM buffer (0.5M KCl, 0.15M CaCl2, 0.25M MgCl2). Add DNA, then flick to mix contents. Incubate on ice for 15 minutes, then heat shock at 42˚C for 90 seconds. Add 100µL SOC media and allow cells to recover for 1 hour at 37˚C. Plate cells on LB plate supplemented with the appropriate antibiotic.
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