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Applications of BBa_M50087

Our novel device consists of two different DNA constructs, which will be transformed into E. coli using two plasmids. The first construct is the sensor -- the ArsS/ArsR TCS, which will constitutively produce the two proteins that will enable the cell to sense acidic pH. The second construct is an actuator that consists of the pUreA promoter and Comet GFP. Under acidic conditions, this will produce the reporter protein upon binding of the phosphorylated ArsR to pUreA.

1.1 ArsS/ArsR TCS

The sensor construct is configured as a two-gene operon. It begins with an inducible promoter, followed by a strong ribosome binding site (RBS), then the ArsS gene. ArsS is then followed by another strong RBS, then the ArsR gene, and finally a terminator. It will be cloned into plasmid number pD441-SR, which has a high copy number and the gene for kanamycin resistance.The high copy ORI ensures that the ArsRS genes are inserted into the E. coli. The kanamycin resistance marker allows us to select for bacteria containing the plasmids to survive. The promoter is a T5 (IPTG-inducible) promoter sourced from the iGEM Parts Registry with part number BBa_50075. It is a standard promoter for bacterial expression and allows us to investigate the dynamic range of ArsS/ArsR gene activity through selective control over gene expression. For both ArsS and ArsR, the ribosome binding site is a strong RBS sourced from the iGEM Parts Registry with part number BBa_50080. A strong RBS was chosen to maximize translation initiation and, thus, produce a larger quantity of these proteins in order to maximize the pH sensitivity of our device. The H. pylori ArsS and ArsR gene sequences were both obtained using UniProt and then optimized for use in E. coli using Integrated DNA Technologies’ Codon Optimization Tool. The codes in the UniProt databases for ArsS and ArsR are A0A1U9ISA2 and A0A1U9IS85, respectively. We added a FLAG tag to ArsS and a histidine (6xHis) tag to ArsR in order to guarantee that we can track the expression of these proteins through assays such as Western blots or enzyme-linked immunosorbent assays (ELISAs). In addition, neither gene sequence had a stop codon, so we added the stop codon “TAA” to both sequences. The optimized and modified sequences for ArsS and ArsR are in the iGEM Parts Registry under numbers BBa_M50085 and BBa_M50086, respectively. The terminator is a T7 Phage terminator, a standard terminator for bacterial expression, sourced from the iGEM Parts Registry. It is listed under parts number BBa_50060.

1.2 Comet GFP Actuator The actuator construct consists of the H. pylori pUreA promoter, a strong RBS, the Comet GFP gene, a low copy ORI, an ampicillin (amp) resistance gene, and a T7 Phage terminator (Figure 4). The pUreA promoter is, as mentioned before, a promoter for the urease gene cluster in H. pylori and is activated when bound by phosphorylated ArsR. The gene sequence for the promoter was sourced from a paper titled “Activation of Helicobacter pylori ureA promoter by a hybrid Escherichia coli-H. pylori ropD gene in E. coli,” published in November 1999 in Gene.5 No modifications were made to this promoter as it is known to be integral to the acid response system in H. pylori, and we want to maximize the likelihood that the TCS and promoter function in E. coli as well. As in the sensor construct, the RBS in the actuator construct is the same strong RBS sourced from the iGEM Parts Registry. The Comet GFP gene sequence is a standard reporter protein which was likewise sourced from the iGEM Parts Registry with part number BBa_K1429000. As the sequence already included a stop codon, no modifications were made to the sequence. Finally, the terminator for this actuator is identical to that used in the above sensor construct, the T7 Phage terminator, again sourced from the iGEM Parts Registry.

2. Bacterial Culture and Transformation We finalized the designs for our sensor and actuator constructs and ordered both from DNA 2.0. We received our sensor construct in powdered form and our actuator construct as an agar stab. Overnight cultures of the sensor construct were then grown and made into chemically competent cells. The actuator construct was grown in an overnight culture using the agar stab to inoculate LB + amp media and purified. The purified actuator plasmid was then transformed into the chemically competent cells with the sensor construct. The transformed cells grew as single colonies on a LB + amp + kan plate. Liquid cultures with cells containing both constructs and grown in LB + amp + kan media were made into a glycerol stock and stored in a -80oC freezer for future use. The LB + amp + kan agar plate was prepared by mixing 100 uL of LB media and 20 uL of 1000x kanamycin and spreading the solution on a LB + amp plate using sterile beads. The LB + amp + kan liquid media was prepared by adding 1 uL of 1000x kanamycin for every 1 mL of LB + amp liquid media.

3. GFP Assay

3.1 pH Dynamic Range GFP Assay To determine the optimal pH that our device can sense, we performed a pH GFP assay that measures fluorescence from a range of 3.5-8.5, 0.1. E. coli that had been double transformed with the sensor and actuator constructs were placed in LB + kan + amp media with 1.0mM IPTG. We selected this high concentration of IPTG to ensure a high enough expression of the ArsS/ArsR sensor system in our cells. Additionally, we created a negative control by placing E. coli containing only the actuator construct in LB + amp media with 1.0mM IPTG. We also created additional negative controls by inoculating both the double- and single-transformed E. coli in LB + kan + amp and LB + amp media, respectively, without any IPTG to ensure that expression of the ArsS/ArsR sensor system would not be induced. We placed all of these liquid cultures in the 37ºC incubator shaker overnight. We made solutions of EZ-Rich media with pHs between 3.5 and 8.5,0.1, at increments of 0.5. We used EZ-Rich media because, compared to LB media, it contributes less noise to the GFP fluorescence readings. Beginning with the EZ-Rich media, we added hydrochloric acid (HCL) dropwise to the media until the desired acidic pH was reached. After making all of the acidic solutions, we began again with pure EZ-Rich media and added sodium hydroxide (NaOH) dropwise to create solutions with the desired basic pHs. The following day, we measured the OD600 of each culture. We took a 1000 µL aliquot from each culture, pelleted the cells, and vacuumed off the supernatant. We then resuspended the cells in EZ-Rich media, such that each aliquot had a final OD600 of 0.2. We then performed a GFP assay following the general protocol outlined in the Experimental Design and Experimental Measurement protocols in the BIOE44 Course Reader and Lab Manual, adhering to the plate set-up seen in Figure 1. Over the course of ten hours, the OD600 as well as the fluorescence at 515nm for each well was measured using the plate reader. Plots of OD600, fluorescence, and normalized GFP fluorescence over time were generated.

In Figure 2 you can see the fluorescence E. coli exhibited over a 10 hour period at varying pHs. From this graph, we concluded that a pH of 6.0 induced the best fluorescence, however, the next best fluorescences are at pH’s of 8.0 and 8.5 . This contradicts our initial hypothesis that GFP will only be expressed in acidic conditions. Though all other pH’s cause E. coli to express some fluorescence (with pH of 3.5 as an exception; this pH was likely too low for the cell’s survival), it is clear that pHs of 6.0 and 8.0 induce a significant response. As mentioned above, we made multiple controls to ensure these results are not due to “leaky” expression. Figure 3 shows the fluorescence exhibited by these controls. Besides the line representing -IPTG at pH of 3.5, most are fairly close to the fluorescence of just the media. We believe the well containing the -IPTG/pH = 3.5 was contaminated, as the graph varies substantially, while all other controls show a smooth trend line. Our control of just the actuator at pH of 8.0 shows that there is indeed leakiness in our system, however, the peak fluorescence exhibited by our experimental well at pH of 6.0 shows nearly double the fluorescence compared to the control (Fig. 2), so we can still conclude that our system exhibits significant fluorescence when in a pH of 6.0.

3.2 IPTG Dynamic Range GFP Assay Having determined that our pH sensor construct produces maximal fluorescence at pH 6.0, we decided to perform another dynamic range experiment to determine the optimal IPTG concentration for sufficient and full induction of ArsS/ArsR expression by performing a GFP assay measuring fluorescence from 0.05 mM to 3.2 mM IPTG. E. coli that had been double transformed with the sensor and actuator constructs were placed in LB + kan + amp media with no IPTG to ensure that no expression of the ArsS/ArsR system was induced before the assay began. Additionally, we created a negative control by placing E. coli containing only the actuator construct in LB + amp media without IPTG, as those cells are unable to express the ArsS/ArsR genes at all. We placed these liquid cultures in the 37ºC incubator shaker overnight. The following day, we measured the OD600 of each culture. We took a 1000 µL aliquot from each culture, pelleted the cells, and vacuumed off the supernatant. We then resuspended each aliquot in pH 6.0 EZ-Rich media such that each aliquot had a final OD600 of 0.2. Again, we used EZ-Rich media because, compared to LB media, it contributes less noise to the GFP fluorescence readings.We then performed a GFP assay following the general protocol outlined in the Experimental Design and Experimental Measurement protocols in the BIOE44 Course Reader and Lab Manual, adhering to the plate set-up seen in Figure 2. Over the course of ten hours, the OD600 as well as the fluorescence at 515nm for each well was measured using the plate reader. Plots of OD600, fluorescence, and normalized GFP fluorescence over time were generated. The results from this experiment were underwhelming compared to our first GFP assay. The maximum fluorescence exhibited by both the double transformed and actuator E. coli, is around 25 000 (Figs. 4 and 5). This is significantly smaller than the fluorescence seen in our first GFP assay at a pH of 6.0 (Fig. 3). In fact, both the experimental and control group show almost identical fluorescence, so, either the system is leaky, making our results in the first GFP assay questionable, or to see significant fluorescence, one must grow E. coli overnight with IPTG before placing it in acidic conditions. This second hypothesis seems more likely, as we grew E. coli overnight in IPTG before we conducted the first GFP assay (varying pHs). However, it would be preferable to conduct both experiments again before making concrete conclusions.

In conclusion: Further testing is needed to ensure the composite part functions at a statistically significant level. Preliminary results indicate that if the E. coli is induced with IPTG overnight, and put in a solution of pH = 6, then significant GFP fluorescence will be observed.

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