Part:BBa_M36016:Experience

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Applications of BBa_M36016

This part is intended to produce a visualizable chromoprotein in the presence of thiosulfate as part of a two-plasmid device system working with a Thiosulfate sensor part (BBa_M36056). This system has a potential application as a noninvasive alternative to colonoscopy in the diagnosis of ulcerative colitis. Ulcerative colitis is associated with higher thiosulfate levels because thiosulfate is an intermediate in the breakdown of hydrogen sulfide, a known indicator of gut inflammation. By using VixenPurple chromoprotein as a reporter, this device has the potential to make an easy diagnostic tool because theoretically the color would be visible for medical screening or home use. Although this device was designed as a proof of concept only, ideally a patient could swallow a probiotic containing bacteria engineered with this system in order to detect gut inflammation without requiring a colonoscopy.

After optimizing the system to work in vitro, we could then optimize this system to work in an E. coli strain that is native to the gut microbiome and conduct in vivo assays in mouse models of intestinal colitis. This would move us one step closer to creating a synthetic, non-invasive, and rapid method of ulcerative colitis diagnosis.

User Reviews

UNIQf5cc73458c3049e0-partinfo-00000000-QINU UNIQf5cc73458c3049e0-partinfo-00000001-QINU

Before testing our device, we performed a dose-response experiment using a stored sample of VixenPurple-producing E. coli cells in increasing concentrations of IPTG inducer. This was necessary to characterize the VixenPurple absorption spectrum because DNA2.0 does not list the single absorption maxima for their chromoproteins. The absorption peak measured was at 580nm. Because this value is difficult to distinguish from the OD600 peak, we used OD700 as our cell density measurement.

Next we performed a functionality test with a dynamic range experiment. We grew cells inoculated from our dual plasmid glycerol stock cells in EZ media with 1000 µM rhamnose and 1000 µM IPTG to induce the pThsRR and pThsS promoters. Samples were grown in triplicate on a deep-well plate (96-well) at thiosulfate concentrations ranging from 1µM-10mM increasing by factors of ten. As negative controls, we used cells grown without thiosulfate as well as cells without with thiosulfate (10mM) but without inducers. Because 10mM is far above the normal thiosulfate concentrations in the body and has been previously shown to saturate this type of thiosulfate sensor, the 10mM was a positive control. As an additional positive control we used pMystery cells which were known to successfully produce VixenPurple chromoprotein when induced with IPTG. The first iteration of this dose-response experiment failed, with both experimental samples and positive controls failing to produce chromoprotein. Thus, we repeated the experiment using new inducers and a modified protocol in which cell concentration was increased from OD0.001 to OD0.2 for growth in the deep-well plates. Normally, dilution to OD0.001 is done to optimize for growth, however, because thiosulfate could be impacting the growth ability of own cells we increased concentration to optimize for protein production.

Figure 1 shows the effect of log-fold increases in thiosulfate dose on chromoprotein expression. While running this experiment, the goal was to visually see purple chromoprotein as an indication that the system was functioning, and to then quantify the expression using spectrophotometry. Given that the dual plasmid system is activated with thiosulfate expression, if the system were to work, we expected to see increases in chromoprotein concentrations as we increased thiosulfate dose. However, no colour was visible after performing the experiment, making it difficult to establish whether or not this relationship was the case. However, we did run the samples through the plate reader, and the above is a synthesis of those results. Triplicates were performed for each thiosulfate concentration, and the error bars illustrate the standard deviations for each condition group. Although the graph itself is downward sloping with increases in thiosulfate concentration, the error bars indicate that this drop is not statistically significant.

As we were having trouble visualizing the chromoprotein, one concern we had was that the thiosulfate itself could be interfering with protein folding. As a result, we grew a strain of bacteria with pMystery – a plasmid that produces a similar purple chromoprotein via addition of IPTG – with thiosulfate to ensure that this was not the case. Figure 2 shows the levels of purple chromoprotein (as measured with OD580) expression of pMystery and the dual plasmid system using three different groups: thiosulfate without inducers, inducers without thiosulfate, and with the addition of both thiosulfate and inducers. The expressions were normalized to ensure that variance in the amount of bacteria was not interfering with the data. Figure 5 confirms that our dual plasmid is not functioning as desired, since no significant differences in chromoprotein expression can be seen between groups. However, we were able to confirm that thiosulfate is unlikely to be interfering with the folding of the chromoprotein, as pMystery both visually and quantitatively produced purple protein pigment with the addition of inducers, regardless of the presence of thiosulfate. In fact, for pMystery, there is no statistically significant difference between the two groups with inducers, which implied that thiosulfate has no effect on pMystery’s function.

Since our functionality/titration assay failed, our next experiment was to check for our proteins using SDS-Polyacrylamide gel electrophoresis and Western blotting using antibodies against the FLAG tag. As a negative control, we used uninduced E. coli cells.

Figure 3 shows the results of the Western with bands at 23.1 kD, corresponding to the expected size of the thiosulfate receptor (ThsR). Samples without inducers did not have visible bands, while relatively strong bands in that region can be seen for samples with added inducer.

Overall, the Western Blot indicated that the plasmid was translating our desired protein; however, there appears to be an issue with the functionality of the overall system. Next steps include fixing and staining bacteria to visualize protein localization and repeating the experiment without tags to check if the tags are inhibiting functionality. A future experiment to perform once functionality is achieved is to characterize chromoprotein stability over time.

Discussion
We think this system failed due to problems with the sensor system rather than this reporter. Because the pMystery chromoprotein was still expressing under these experimental conditions, a problem with the reporter system was unlikely. As indicated above, the western blot showed that both ThsS and ThsR were being expressed with IPTG and rhamnose present. This indicated that our system is probably not working due to sensor protein dysfunctionality, because the actuator should be working under these conditions and both sensor proteins are being expressed.

After getting this new data, we first went to verify that our DNA design was correct. After extensive review of the pThsS and pThsRR files in benchling, we determined that the lack of protein expression was not due to errors in designing our plasmids. The ORF sequences for our desired proteins exactly matched the codon optimized versions from the Tabor lab, except for the FLAG and His tags. This leads us to the next hypothesis that the FLAG or His tags may be interfering with proper protein function. There could be multiple different causes of this dysfunction. First, the C-terminal His tag on ThsS may have interfered with membrane localization. If the protein was unable to localize at the membrane, this would explain our system’s malfunction because the protein would not be able to sense the extracellular thiosulfate. To test for this, we could fix a sample of E. coli and stain for the ThsS protein. We could then visualize the samples via confocal microscopy to see if the protein is localizing to the membrane.

It is also possible that the tags interfered with the interactions between (1) the thiosulfate and ThsS, (2) between ThsS and ThsR, or (3) between ThsR and the PphsA342 promoter/RNA polymerase. Our searches were unable to identify any solved structures of ThsS and ThsR, so it is difficult to immediately determine if our tags are hindering any protein interactions. One follow up experiment might express these proteins without His and FLAG tags, and if we do see chromoprotein expression, this indicates that the tags were interfering with protein interactions. Furthermore, we could use the CRISPR system to selectively induce mutations at the C-terminus of each protein, since this is where we added our tags. This could give us important information about which regions of each protein participate in different interactions.

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