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Applications of BBa_K3558000

The iGEM21_UNSW_Australia team is a Phase II project continuing the work of iGEM20_UNSW_Australia. The 2020 UNSW Phase 1 team had successfully transformed and expressed HSP22E in E. coli BL21 (DE3). To continue this effort, our team had designed assays to test the growth and survival of E. coli BL21 expressing the HSP22E protein.

Our wet lab goal for this year was to ascertain whether expression of heat shock protein HSP22E would increase thermotolerance of E. coli. To do this, we tested the effects of E.coli survival at extreme temperatures using a survival assay, and its growth at elevated temperatures using a growth assay. For the growth assay, it was hypothesised that the presence of HSP22E would allow for improved growth at high temperatures compared to the optimal growth temperature. Whilst for the survival assay, it was hypothesised that the presence of HSP22E would result in increased survivability of E. coli at higher temperatures.

Our assays used 37ᵒC, the optimal growth temperature for E. coli, as the control temperature for testing against elevated temperatures (Doyle and Schoeni, 1984). We used E. coli expressing mCerulean3, a fluorescent protein which would not confer thermotolerance, as the control for our testing since it has a molecular weight of 26.8kDa, similar to that of HSP22E (25.2kDa) (Markwardt et al., 2011; Watkins et al., 2013). As protein overexpression would create stress for cells and affect cell growth, including a control overexpressing mCerulean3 was important to ensure that fair comparison was made.

Another critical aspect was the molecule IPTG for inducing protein expression. The control - mCerulean3 and HSP22E samples were expressed in the pET19 plasmid which uses IPTG to induce protein expression (Kroemer n.d.). Included in both assays were samples of HSP22E and mCerulean3 that did not have IPTG added. As an additional control measure, we included samples without IPTG added to ensure that affected thermotolerance in the growth and survival assays were due to the expression of HSP22E.

Unfortunately, due to COVID-19 restrictions our wet lab team only completed preliminary testing of our assays. However, we have used this initial data to refine the assay design for future testing. Additional optimisation will be needed in future experiments to finalise more accurate and robust assay designs for growth and survival.

Growth Assay of BBa_K3558000

The Growth assay aimed to test if the presence of HSP22E increased the thermotolerance of E. coli and allowed for growth at temperatures higher than optimal. Unfortunately, due to limited laboratory access resulting from COVID-19 restrictions and this assay was performed once with no repeats.

Method:
A total of four samples were tested: HSP22E with IPTG, HSP22E without IPTG, mCerulean3 with IPTG, and mCerulean3 without IPTG as seen in Figure 1.

Figure 1: Flow diagram of heat shock protein growth assay method. Image created in BioRender.

Overnight cultures of E. coli containing mCerulean3 (control) and HSP22E genes were prepared. Day cultures were then inoculated using the overnight cultures and were allowed to grow to an optical density (OD600) of 0.6. IPTG was added to induce protein expression. All sample cultures were subsequently diluted to an OD600 of 0.1 with Luria-Bertani (LB) broth containing ampicillin. The samples were incubated in shaking incubators of 37° C and 45° C. The OD600 of the cultures was measured using a spectrophotometer every hour for six hours.

The OD600 measurements were adjusted to E. coli cells per mL using the standard of 1.0 OD600 unit is equal to 8x108 cells per mL.

Results:
Results of the growth assay are presented in Figure 2.

Figure 2: Growth curve of E. coli cells per mL at 37ᵒC (A) and 45ᵒC (B) over approximately six hours. Y-axis represents the time measured in minutes, while x-axis represents the number of cells per mL.

Minimal differences were observed among the four samples incubated at 37ᵒC and 45ᵒC. Interestingly, samples at 45ᵒC had greater cell numbers than samples at 37ᵒC at time point 70 minutes. Overall, E. coli incubated at 45ᵒC had lower cell counts compared to the samples incubated at 37ᵒC throughout the experiment.

Discussion:
Our experiment made several discoveries that were subsequently used to improve the assay design.

It was hypothesised that the samples expressing HSP22E would grow more efficiently and faster at 45ᵒC than the control samples that lack HSP22E. However, this effect was not observed. At 45ᵒC mCerulean3, the control, and HSP22E, the target, had minimal differences in growth. The presence of IPTG had no observable impact on the growth of the samples. The lack of differences between the “with” and “without” IPTG samples indicates that IPTG may have not induced expression in the “with IPTG” samples. Without confirmation of our target, HSP22E, being present, the assay could not successfully assess if HSP22E impacted growth at elevated temperatures.

To continue the assay in future it will be necessary to perform a protein extraction of the samples to ensure that both the target and control proteins are being successfully induced by the addition of IPTG. Although our hypothesis was not confirmed we did gain valuable insight into the assay design that can be applied for improvement in future experiments.

The aim of the assay was to assess if HSP22E could support growth under heat stress at elevated temperatures, however the temperatures tested did not indicate heat stress was occuring. It was hypothesised that the enhanced thermotolerant sample, HSP22E with IPTG, would have greater growth and other samples would have minimal to no growth at 45ᵒC. This did not occur, and at 45ᵒC all samples had the same growth curve and minimally reduced cell counts, compared to the control temperature. It was considered from the results that 45ᵒC may not be effectively causing heat stress on the cells. This would imply that the presence of HSP22E would have no effect on growth, making the assay unsuccessful in testing the aim. These results indicated investigation into the suitability of testing at at 45ᵒC was needed to improving the design of the growth assay.

To develop a robust assay that assessed the effect of HSP22E on thermotolerance, potential causes for the previously discussed results were investigated. In addition, the team consulted our lab mentors to aid in troubleshooting our design. Through our investigation two potential issues were identified in the assay design. Firstly, there was no calibration or additional temperature readings taken for the incubator used in the 45ᵒC test, this indicated that there was potentially ineffective heating of the samples during the assay. In addition to ineffective heating, 45ᵒC was found to not be a sufficiently high temperature to test the thermotolerance in E. coli. Research has found that E. coli can grow effectively at 45ᵒC and in some cases, growth can be consistent up to 49ᵒC (Fotadar et al., 2005). We subsequently altered our design to assess growth at 51ᵒC to test more effectively test normal and heat-stressed growth of the samples. Additionally, the team sought access to a metal heat block, as it would be a more effective instrument for heat transfer and can reach temperatures for above 45ᵒC. By identifying issues in our assay we adjusted the design to more effectively assess the effects of HSP22E on thermotolerance in E. coli.

Survival Assay of BBa_K3558000

We conducted a survival assay to determine the effect of high temperatures on the survival of E. coli. We hypothesised that E. coli expressing HSP22E would have an enhanced survival rate compared to control samples when subjected to heat shock.

Figure 3: Flow diagram of heat shock protein survival assay method. Image created in BioRender.

Figure 4: Survival assay cell concentration at 37℃ and 46.4℃. HSP22E w IPTG stands for E. coli containing HSP22E gene induced to express the gene with IPTG. Similarly, HSP22E w/o IPTG stands for E. coli containing HSP22E gene without IPTG induction. mC3 w IPTG and mC3 w/o IPTG represent the E. coli samples with the gene for mCerulean3 with and without IPTG induction, respectively.

Figure 5: SDS-PAGE of samples after Bugbuster protein expression. The samples were collected prior to heat shock after 1 hour IPTG induction.

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