Difference between revisions of "Team:Exeter/Results"

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<h2 id="h1">Chassis determination </h2>
 
<h2 id="h1">Chassis determination </h2>
  
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  <p> As we wanted to express modified pili in E. coli we ideally needed a chassis that did not natively produce pili. The genome of <i>E. coli</i> MG1655 is annotated as having the <i> fim operon </i> (Accession NC_000913) and was therefore proposed to act as a positive control in all experiments. For modified pili production two strains were initially proposed: <i>E. coli</i> Top10 (Invitrogen) and <i>E. coli </i> DH5&#945; (NEB).  Top10  is a derivative of DH10B, the genome of which (Accession CP000948) is lacking an annotated <i>fim operon</i> whereas there is no published genome for DH5&#945;. Therefore it was important to verify whether these two strains were lacking the pili producing <i>fim operon</i>.</p>
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  <p> As we wanted to express modified pili in <i>E. coli</i> we ideally needed a chassis that did not natively produce pili. The genome of <i>E. coli</i> MG1655 is annotated as having the <i> fim operon </i> (Accession NC_000913) and was therefore proposed to act as a positive control in all experiments. For modified pili production two strains were initially proposed: <i>E. coli</i> Top10 (Invitrogen) and <i>E. coli </i> DH5&#945; (NEB).  Top10  is a derivative of DH10B, the genome of which (Accession CP000948) is lacking an annotated <i>fim operon</i> whereas there is no published genome for DH5&#945;. Therefore it was important to verify whether these two strains were lacking the pili producing <i>fim operon</i>.</p>
  
  

Revision as of 19:20, 1 November 2017

Results

Chassis determination

As we wanted to express modified pili in E. coli we ideally needed a chassis that did not natively produce pili. The genome of E. coli MG1655 is annotated as having the fim operon (Accession NC_000913) and was therefore proposed to act as a positive control in all experiments. For modified pili production two strains were initially proposed: E. coli Top10 (Invitrogen) and E. coli DH5α (NEB). Top10 is a derivative of DH10B, the genome of which (Accession CP000948) is lacking an annotated fim operon whereas there is no published genome for DH5α. Therefore it was important to verify whether these two strains were lacking the pili producing fim operon.

Using a genomic extraction and PCR we attempted to isolate and amplify three known genes of the fim operon in MG1655 and DH5α. We ran the products of these two preparations down an agarose gel. The results of the electrophoresis procedure showed the same bands in the MG1655 as in the DH5α, representative of three genes in the operon. This demonstrates DH5α's possession of the operon and therefore the unsuitability of DH5α as the chassis for our plasmid inserts.

Figure 1: This gel and the gel in Figure 2 show the genetic analysis of three strains of E. coli: MG1655, DH5α and Top10. In both cases, three known genes of the fim operon have been amplified. The wells 0-8 on this gel contain the following: hyperladder 1, DH5α FimD, MG1655 FimD, Negative control (water) FimD, DH5α FimH, MG1655 FimH, Negative FimH, DH5α FimA, MG1655 FimA, Negative FimA.
Figure 2: The wells in this gel contain the following, 0-9: hyperladder 1, Top10 FimA, Top10 FimD, Top10 FimH, MG1655 FimA, Negative control(water) FimD, MG1655 FimH, Negative FimA, MG1655 FimD and negative control FimH. Evidently, MG1655 and DH5α contain the three genes and therefore produce pili natively. Meanwhile Top10 lacks all three and so does not produce pili.

The other strain identified in the literature as potentially lacking the fim operon was Top10. A similar procedure was used as above, involving a genomic extraction and an enzymatic amplification of the genes of interest. Once more, the MG1655 strain was used as a positive control. The PCR products were run down an agarose gel and the results showed that Top10 does not contain the three genes of interest and so does not have the operon or natively produce pili.

Overall, these results confirmed that MG1655 would be ideal as a positive pili producing control and, that Top10 would be a suitable chassis for our modified pili whereas DH5α would not. In addition two further chassis strains were chosen, the E. coli K-12 FimB knockout strain (JW4275-1 from the Keio collection Baba et al) that the Harvard 2015 iGEM team demonstrated did not produce pili and the E. coli K12 FimH knockout strain (JW4283-3 from the Keio collection Baba et al) which is deficient in the gene that we specifically wanted to modify. Finally, E. coli BL21(DE3) was used to allow initial protein expression to be investigated using the T7 promoter.

Modified Pili Expression

Part construction

The modular cloning strategy (described in the materials and methods) was used an attempt to build plasmids containing either: the fim operon under two different promoters; or six modified versions of fimH (and wild type fimH) under for different promoters. The results of our cloning attempts are given in Table 1.

Coding sequence P_T7 P_Rha P_Ara P_T5 P_J23100
FimH Yes N/A
FimH_1_His Yes N/A
FimH_1_SynMT Yes N/A
FimH_1_MouseMT Yes N/A
FimH_1_Plastocyanin N/A
FimH_1_sfGFP Yes N/A
FimH_225_sfGFP Yes Yes N/A
FimH_258_sfGFP Yes N/A
Fim operon N/A N/A Yes N/A Yes

Table 1: Combinations of Promoters and FimH fusion proteins for which cloning was attempted

Part testing

FimH_sfGFP expression

Our new part BBa_K2324011 , the FimH_225sGFP under control of the T7 promoter was induced and examined for fluorescence. The T7 promoter would give very strong expression and sfGFP would both give a visual indication of successful expression and folding. As a large protein, sfGFP would push the chaperone-usher pathway to its steric limits. Overall, the FimH_sfGFP fusions would tell whether we could put large proteins on FimH and still maintain robust export and pilus biogenesis.

Figure 3 This graph outlines the difference in fluorescence at a wavelength range of 477-515 nm between a sample of wild type BL21(DE3) and BL21(DE3) transformed with the T7_FimH_225_sfGFP construct. According to standard curves made in the lab on the same equipment and settings, we produced a 130 ng/ml concentration of sfGFP in the sample.
Figure 4: These data from an image stream show the fluorescence profile for wild-type (left) BL21(DE3) and BL21(DE3) with T7_FimH_225sfGFP (right).The wild type demonstrates no significant fluorescence, while same cell with the single plasmid insert shows a strong fluorescent signal in the highlighted portion of cells.

These results show that a number of cells in the overall culture produced strong fluorescence. This fluorescence suggests successful folding of the sfGFP which can be taken as evidence by proxy of FimH folding. The result also suggests that sfGFP is able to move through the pore formed during pilus biosynthesis. Moving on from this result, we were confident that the fusion of metal binding domains to the FimH protein would not negatively affect pilus formation as our chosen domains, and other potentials that may make up a modular toolkit, were smaller than sfGFP .

Figure 5 : Western blot of BL21 WT and BL21 with our T7-FimH_225_GFP construct, 1-5: sfGFP, BL21 WT soluble fraction, BL21 WT pellet, BL21 with T7-FimH_225_GFP solluble fraction, BL21 with T7-FimH_225_GFP pellet

Another way of testing the sfGFP parts was immunogold labelling. This sequence specific method would ideally tell us whether the cell was exporting our modified pili, or just the native pili. It was carried out in the BL21(DE3) strain.

Figure 6 Pictured here are two wild type BL21(DE3) cells which should not contain the sfGFP antigen in their FimH proteins. A few gold particles are visible in these micrographs, though they can likely be attributed to non-specific binding.
Figure 6 In contrast, when BL21(DE3) in transformed with T7_FimH_225_sfGFP, it appears to specifically bind the gold particles as shown. The images suggest successful expression and export of these modified FimH proteins and specific binding. On the image on the right, the gold particles appear to align with the pili on the cell surface.

We were unable to detect fluorescence in cells that had the FimH fusion protein under control of the rhamnose inducible promoter despite experimenting with the induction protocol over a range of growth temperatures and rhamnose concentrations . Given the relatively low proportion of cells in the overall culture of the T7_FimH_225_sfGFP that showed significant fluorescence, it is perhaps unsurprising that a weaker and less well characterised promoter(than T7) should not give strong results. Our consequent decision was to attempt to test the rhamnose inducible promoter by other means. We used a fusion of 6xhistine tag and FimH as an alternative reporter mechanism with the aim of establishing whether modified FimH constructs could be expressed with P_Rha.

FimH_1_His expression

We therefore looked to demonstrate expression of the FimH_1His protein in the pili of our cells. This part was designed as a reporter to tell us whether we could modify the FimH protein directly at the N-terminus position, after the point of signal peptide cleavage.

The results of an SDS-PAGE and subsequent Western blot provided bands at the corresponding molecular weights for the FimH_1His protein which also had binding affinities for an anti-His antibody. The result of a band that corresponds to a molecular weight marginally lighter than the entire intact FimH_1His evidentiated expression of the protein, and suggested that the signal peptide had been cleaved upon the proteins delivery to the cell surface membrane. This makes a case for successful export of the protein and therefore successful pilus biogenesis.

Figure 7 The wells of the polyacrylamide gel contained the following, 1-10:marker,ΔFimH_1His pellet, ΔFimB_1His pellet, Top10_1His pellet, ΔFimH_1His pellet, ΔFimH_1His supernatant, Top10_1His supernatant, BL21(DE3)_1His supernatant, ΔFimB_1His supernatant and marker. The band of importance is the one at 28-30kDa which represents the FimH protein, potentially having been exported and had its signal peptide cleaved.
Figure 8 The wells is this gel are filled, 0-9, as follows: marker, BL21(DE3)WT pellet,Top10 WT pellet, ΔFimB WT pellet, ΔFimH WT pellet, BL21(DE3) WT supernatant, Top10 WT supernatant, ΔFimB WT supernatant, ΔFimH WT supernatant and BL21(DE3)_1His pellet.
Figure 9 The columns of the Western blot correspond to the following samples,0-9:marker,ΔFimB_1His pellet, Top10_1His pellet,BL21(DE3)_1His pellet,BL21(DE3)_1His pellet,BL21(DE3)_1His supernatant, ΔFimH_1His supernatant, ΔFimB_1His supernatant, Top10_1His supernatant and marker. The bands present here demonstrate the affinity of protein with the same molecular weight as FimH with anti-His antibodies. This would imply that FimH_1His is being expressed

*INSERT IMAGES*

Fim Operon expression

Figure 10 On the right is an electron micrograph of an E. coli MG1655 cell. Pili are clearly visible on the surface of the cell. On the left is an image of Top10, which displays no pili.
Figure 11 Top10 with the fim operon under the control of promoter P_J23100 (left) and P_Ara(right), showed no visible signs of pili expression with insertion of the operon alone.
Figure 12 ΔFimB should not, theoretically, produce pili. The regulatory gene FimB has been knocked out, and so the operon has effectively been switched off. These electron micrographs show wild type ΔFimB with strong, peritrichous flagellar expression, but no visible signs of pili. The image on the right shows little evidence of pili or flagella connected to the cell surface, which suggests that the negative staining technique can be damaging to these structures and could cause detachment and fragmentation.
Figure 13 We transformed a plasmid containing the fim operon under control of promoters P_J23100(left) and P_Ara(right). Both exhibit flagella on their cell surface, but the right electron micrograph shows a suggestion of pili. This suggests that the P_Ara construct was successful in synthesising pili (minus FimH).

Applied Design

Hydrocyclone

To see how effective our hydrocyclone was at separating sediment from a contaminated solution, follow this link to our hydrocyclone page, Hydrocyclone Results

Metal Binding Reactor

Biosecurity

To see that results we obtained for how the percentage of bacteria change with exposure time to UV light, follow this link to our applied design page, UV results