Difference between revisions of "Part:BBa K3558000:Experience"

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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

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

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.

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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