Team:Peking/KillingImprovements

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Introduction

In our project, we eliminate algal bloom by designing E. coli to secrete proteins that can lyse the cyanobacteria. To make our project feasible in future applications, we have to find ways to enhance the killing effect. As our model indicates, binding our E. coli to the cyanobacteria can help us achieve this goal. So we searched for proteins that can bind to the targeted M. aeruginosa cells and we chose to work with microvirin (MVN), a mannan binding lectin in Microcystis aeruginosa PCC7806, since of its high specificity to the cyanobacteria and its availability to the E. coli. Microvirin is a protein consisting of 108 amino acid residues and it has one mannan binding site which can bind to the LPS of the M. aeruginosa cells [1] . In our project, MVN was displayed on the surface of E. coli cells using a kind of truncated INP (INPNC). By this way, we could bind our E. coli to M. aeruginosa and thus help us increase the overall killing effect.

However, cyanobacteria are usually floating in the surface water due to its gas vesicles and colony formation in the field. So we also equipped our cells with gas vesicles to deal with the problem that E. coli cells mostly sink to the bottom when put into water. This will increase the opportunity for an E. coli cell to approach a cyanobacteria cell and enhance the killing effect too.

Design

To make our genetically engineered E. coli bind to Microcystis aeruginosa, we used a kind of truncated Ice Nucleation Protein (INPNC) to display microvirin (MVN) on the surface of our E. coli. We also used GFP as a reporter to make the binding effect "visible".

INPNC is a kind of modified INP with truncation of the entire internal repeating domain. We chose to work with INPNC because it has been reported to be an excellent surface display system used in E. coli in recent years [2,3]. INPNC can be be expressed on the cell surface at a high level, without affecting the cell membrane structure and cell proliferation, which is really crucial in our case. Also, the size and structure of MVN are both proper for the INP display system [2].

The plasmid containing INPNC coding sequence that we used came from the iGEM distribution 2014 (BBa_K523013).

Figure 1. Schema of the surface display system using INP and INPNC. INP N- domain, which is relatively hydrophobic, anchors INP in the OM (outer membrane). INP C-domain is hydrophilic and exposed to the medium, which can be used to link the passenger protein on its C-terminus. The internal repeating domains serve as the template for ice nucleation, and are not necessary for protein display. Depicted are the (a) full- length INP with no truncation and (b) INP with truncation of the internal repeating domains.

MVN is a lectin isolated from the cyanobacterium Microcystis aeruginosa PCC7806. It is an 108 aa protein that consists of 2 domains. According to a carbohydrate microarray carried out previously, MVN binds to carbohydrate and the highest signals are observed with structures that contain α(1 → 2) linked mannose residues [1]. A binding partner of MVN was identified in the lipopolysaccharide fraction of M. aeruginosa PCC7806 and it possibly represents the O-antigen of a LPS. MVN has been previously expressed in E. coli and its binding effect to cyanobacteria cells is highly species-specific [1]. The availability and specificity are the reasons why we chose MVN in our project.

We optimized the gene sequence of MVN at first and de novo synthesized this gene from commercial company (Genscript, Nanjing, China).

Figure 2. Solution structure of MVN. MVN consists of 2 domains, domain A and domain B, which are colored yellow and blue respectively. Domain A is formed by residues 38-93 while domain B is formed by residues 1-37 and 94-108. The mannan binding site is only found in domain A. This figure is taken from Protein Data Bank (PDB ID: 2Y1S).

We fused INPNC and MVN together to anchor MVN on the outer membrane. MVN is oriented outwards into the medium to recognize and bind to the specific cyanobactera cells. We added a 10 aa flexible protein domain linker in between to make sure that MVN and INPNC can function separately without much mutual interference. The fusion protein INPNC-MVN is expressed under a library of constitutive promoters including BBa_J23106, BBa_J23105, BBa_J23114, BBa_J23117 and BBa_J23113. We also designed our E. coli to express GFP at the same time. The E. coli cells were later co-incubated with M. aeruginosa and observed under the fluorescence microscope afterwards. The fluorescence of GFP and chlorophyll A can help mark our E. coli and M. aeruginosa so that we could identify the binding effect later.

Figure 3. Schematic representation of the binding reaction between E. coli and Microcystis aeruginosa. INPNC anchors at the outer membrane of the E. coli and displays MVN on its C terminus. Linker1 is the blue strand between INP C-domain and MVN . MVN has one Man Α(1 → 2) Man binding site which is a typical component in the LPS of the M. aeruginosa. E. coli and M. aeruginosa are bound together by the intermolecular force.

In addition to this E. coli - M. aeruginosa binding assay, we also designed two additional tests. One test is a direct binding assay with M. aeruginosa and purified MVN protein to prove the binding ability of MVN directly and the other test is a rTEV protease digestion assay to estimate the amount of MVN oriented outwards. Detailed information will be described in the Results.

Results

MVN - M. aeruginosa binding assay

We carried out a binding assay using purified MVN protein and M. aeruginosa to test the binding ability of the MVN produced by our E. coli. A similar MVN binding assay has been carried out by Kehr, J. C. et al. in 2006, but our experiment is different in that we fused eYFP to the MVN and we did the experiment with two different strains of cyanobacteria.

We used pET-21a(+) as the vector to construct a plasmid for the expression of the His6-tagged version of MVN-eYFP (see Fig. 4) During construction, we retained the promoter, RBS, His&#8901Tag and terminator that were on the original vector. We inserted MVN and eYFP coding sequence into the plasmid via Gibson Assembly. After the construction, the plasmid was transformed into E. coli BL21(DE3) cells and induced afterwards to initiate the expression. After lysing the cells, the crude extracts were applied to Ni2+-nitriloacetate columns and the fusion protein was purified and later used in our binding assay.

Figure 4. Final construct of the protein-purifying plasmid. The promoter, RBS and terminator are native ones on the pET-21a(+) vector. Linker1 is the 10aa flexible protein domain linker added to avoid protein interference between MVN and eYFP.

The purified protein was later used in binding assay with the cyanobacteria M. aeruginosa FACHB-1343. The cyanobacteria colony were broken up by mild sonication at first. The protein was added to aliquots of cyanobacteria culture and later observed under fluorescence microscope. The detailed protocol of protein purification and binding assay can be found in the Protocols.

The fusion protein was designed to be MVN-mRFP at first instead of MVN-eYFP. We finished the purification of MVN-mRFP (see Fig. 5) this summer it was used for testing afterwards. However, our fluorescence microscope was not fine enough to distinguish the fluorescence of mRFP and chlorophyll A. We changed the fluorescence protein to eYFP and the results will be available by the Giant Jamboree.

Figure 5. Purification of MVN-mRFP. MVN-mRFP was induced in E. coli BL21(DE3) using 1mM IPTG. Coomassie blue stained SDS-PAGE shows the finished purification of MVN-mRFP via Ni2+-nitriloacetate column. The arrow in the figure indicates proteins corresponding to the molecular weight of MVN-mRFP (38kDa).

rTEV protease digestion assay

INP can help display our protein on the outer membrane, but the orientation is unsure. So we designed this rTEV protease digestion assay to verify that MVN is oriented outwards into the medium. Also we can roughly estimate the amount of outward-displayed MVN that can be digested each E. coli from the result too.

We constructed the functioning plasmid by the means of PCR and Gibson Assembly. The plasmid uses pSB1C3 as the vector and expresses the fusion protein INPNC-MVN-mRFP. We designed a 7aa protein linker named Linker2 between INPNC and MVN which can be specifically recognized and digested by rTEV protease. Also, there is a Linker1 between MVN and mRFP to ensure that the fluorescence is not affected. The coding sequence is regulated by the promoter J23105, the RBS B0034 and the terminator B0015. We transformed the plasmid into E. coli BL21(DE3) after the construction work.

Figure 5. Final construct of the plasmid for rTEV protease digestion assay. Linker2 is a 7aa linker that can be specifically digested by rTEV protease. Linker1 is the 10aa flexible protein domain linker mentioned in Figure.4. The whole INPNC-MVN-mRFP fusion protein is expressed under the constitutive promoter J23105.

To make our results persuasive, we also constructed 3 control groups: C1, C2 and C3. Plasmid C1 has the Linker2 shifted to Linker1 so that it can not be digested by rTEV protease. Plasmid C2 has the INPNC and Linker2 sequence deleted so that MVN-mRFP is not displayed on the cell surface. Plasmid C3 has the whole coding sequence from INPNC to mRFP deleted. We also have a blank control group with no cells added.

Figure 6. Gene circuits of the control groups. (a) Plamsid C1 differs to the original plasmid in that Linker2 is shifted to Linker3, which is a 8aa flexible protein domain linker that cannot be cut by rTEV protease. (b) Plasmid C2 has the coding sequence of INPNC and Linker2 deleted. (c) Plasmid C3 is a plasmid with only the promoter, RBS and terminator left on the vector.

Cells of the experiment group and control groups were cultured to the exponential phase. Afterwards rTEV protease as well as buffer were added to the cell cultures according to the protocol from the company. The cell cultures were co-incubated with rTEV protease and centrifuged afterwards to separate the cells and MVN-mRFP that was cut down by the protease. The fluorescence intensity of the supernatant could stand for the amount of MVN-mRFP and was measured by microplate reader (For experimental details please see the Protocols).

The same procedure was applied to every group for 3 times. The average results are shown in the chart below (see Chart 1), and the fluorescence intensity data is shown in A.

Chart 1. The fluorescence intensity data measured by a microplate reader.

As can be seen from the results shown here, the fluorescence intensity of the experimental group is higher than that of the C2 and C3 groups by a large margin. Considering the precision of our equipment, this can demonstrate that the MVN-mRFP fusion protein is truly displayed outwards by using the INPNC displaying system. The results of the Experimental group and C1 group are close and it may be because of the breakage of Linker1 when we centrifuged the cells. However, the relatively high result of the C1 group is still in accordance with the demonstration.

E. coli - M. aeruginosa binding assay

This assay is the final testing to identify the binding effect of our genetically modified E. coli. First, we designed and constructed the plasmid for the experiment group (See Fig. 7). As described in the Design, we inserted 2 transcription units into the same vector pSB1C3. One transcription unit that encodes INPNC-MVN under a library of promoters was first introduced by the means of PCR and Gibson Assembly. The second transcription unit that codes for GFP only was first constructed in another plasmid and later inserted into the first plasmid by Biobrick Standard Assembly. We also constructed one plasmid for the control group that only had the second transcription unit encoding GFP. After construction, the plasmids were transformed into E. coli BL21(DE3) cells.

>Figure 7. The final construct of the “Binding” plasmid. One transcription unit that expresses INPNC-MVN and one that expresses GFP are inserted into the same plasmid pSB1C3. GFP is expressed under the constitutive promoter BBa_J23117 while INPNC-MVN is expressed under the constitutive promoter J23105. INPNC and MVN are separated by Linker1 which is a 10aa flexible protein domain linker.

We also carried out this binding test with M. aeruginosa FACHB-1343. The cells of the experiment group and control group were both incubated till their OD600 reached 0.6. Then both E. coli and M. aeruginosa were applied to short period of mild sonication to break up the colony formed and harvested by centrifugation and PBS washing. E. coli and M. aeruginosa were co-incubated and observed under fluorescence microscope afterwards. We used microscopic counting to quantify the binding effect.

The binding assay was carried out with both the experimental group and the control group. Each test was done for several times to make the results persuasive. We randomly chose 18 views for both the experimental group and the control goup. Every view was observed in the blue and green channel respectively and later merged together to see the binding effect. The binding effect of the experimental group is shown below in Fig. 9.

>Figure 9. Binding of E. coli to M. aeruginosa observed under the fluorescence microscope. This mosaic is made up of several sectional views taken by the fluorescence microscope. The red dot represents the M. aeruginosa cell and the green rod represents the E. coli cell.

As can be seen from the figure, the binding effect can be observed qualitatively. Later, we used microscopic counting to determine the exact binding ratio. The maximum number of E. coli cells bound to one M. aeruginosa is 4, so we counted the amount of M. aeruginosa cells with 0, 1, 2, 3 and 4 E. coli cells bound. All of the cyanobacteria included in the 18 views were counted, and the result is shown in Fig. 10.

The contrast of binding proportion between the experiment group and control group is notably different. In addition, the difference is even greater when the binding amount reaches 3 or 4, when the random binding is less possible. This result does prove that our design really works and that we can successfully bind our E. coli to M. aeruginosa.

>Figure 10. Quantitative binding effect of E. coli to M. aeruginosa.

Gas vesicles

We succeeded to bind E. coli cells to M. aeruginosa cells, and this will improve our killing effect remarkably as our model indicates. However, it is often mentioned in papers that cyanobacteria are appearing as floating colonies in the field when algal bloom bursts[1]. Also, during our field investigation to Taihu Lake, we observed that cyanobacteria were actually floating in fresh water examples (see Fig. 11). Because of this, floating E. coli is essential for our project, which was a part of the project that the OUC-China iGEM team did in 2012 (https://2012.igem.org/Team:OUC-China/Project/GVP/GasandBackground).

Figure 11. Cyanobacteria are floating in the fresh water example. This picture is taken from the NIGLAS (Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences).

Two proteins, GvpA and GvpC are needed to form a protein complex to help the E.coli cells float. GvpA, a 70 aa hydrophobic protein consisting, is assembled as the framework of the protein complex, which is later stabilized by the hydrophilic protein GvpC, a larger protein consisting of 162 aa.[4] GvpA and GvpC can assemble into a low-density protein cylinder in assistance of floating (see Fig. 12(a)). Gas fills space inside the protein complex by diffusion while water is rejected outside.

We communicated with the OUC-China iGEM team this year, and they helped us confirm the availability of the gas vesicle design. The bacteria did float during the repeated trial and the result is shown in Fig. 12(b). They also kindly provided us with the gene circuit and we also redid the experiment to confirm the viability.

Figure 12. (a) Schematic presentation of the Gas Vesicle formation. Two proteins, GvpA and GvpC, are expressed in E.coli at the same time. Then, they can polymerise in to a protein complex gas vesicle which has a lower density than water so that E.coli can be floating in the water. (b) The E. coli cells were observed to float during the repeated trial by the OUC-China iGEM team.

By equipping the bacteria with gas vesicle, we can help make our project become more feasible in the real ecosystem.

References

1.Kehr, J. C., Zilliges, Y., Springer, A., Disney, M. D., Ratner, D. D., Bouchier, C., ... & Dittmann, E. (2006). A mannan binding lectin is involved in cell-cell attachment in a toxic strain of Microcystis aeruginosa. Molecular microbiology, 59(3), 893-906.

2.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.

3.Lee, S. Y., Choi, J. H., & Xu, Z. (2003). Microbial cell-surface display. Trends in Biotechnology, 21(1), 45- 52.

4.Walsby, A. E. (1994). Gas vesicles. Microbiological reviews, 58(1), 94.

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