Team:Newcastle/Measurement

spacefill

Measurement Award

Rationale and Aim

Assess robustness of a GFP reporter construct in a range of environments.

Background Information

In order for advances in synthetic biology to be successful it is necessary to develop precise and robust standards for measurements of parts and devices. Biological systems are extremely sensitive to cellular and environmental changes, therefore standardisation of gene expression is essential for the reported measurements to be reliable. The lack of these standards presents a potential limitation to the creation of Sensynova and other genetically engineered devices. To address this issue, we examined variability found between identical genetically engineered devices under different environmental conditions and as a result of using different assembly standards.

Optimum DNA Assembly Standards

Gibson assembly proved to be the least time-consuming method to manually produce the synthetic GFP reporter gene compared to PhytoBricks and BioBricks. The production of the GFP reporter construct using the Biobricks method was unsuccessful; the multiple steps involved, time consuming methodology and inefficient enzyme activities throughout this process lead to a limited number of parts that were able to be joined. Additionally, it was found that joining the smaller parts; the promoter and RBS was more challenging than joining the larger parts in the manual assembly. The PhytoBricks assembly method successfully produced the GFP reporter construct however it involved more steps and was more time consuming than Gibson assembly. However, a major limitation of this investigation was human error and a difference in user. The Gibson assembly construct had a higher GFP expression level after 24 hours, at 7.46x105 AFU/OD600 with the standard error of 6.51x104 AFU/OD600, compared to the PhytoBricks assembly construct which had the final GFP expression level of 2.17x105 AFU/OD600 with the standard error of 6.68x104 AFU/OD600. There was a significant difference in the GFP expression between the PhytoBricks and Gibson Assembly constructs (Mann-Whitney, U=483, n=25,25, p<0.05).

Optimum Conditions for Gene Expression

2.1. Growth Media:
The 5-alpha strain containing the synthetic reporter construct which was grown in LB media, at 37ᵒC and a neutral pH, had a higher final GFP expression level, at 6.18x105 AFU/OD600 with the standard error of 1.69x105 AFU/OD600, compared to when grown in SOC media which was 4.08x104 AFU/OD600 with the standard error of 1.76x104 AFU/OD600 at 24 hours. There was a significant difference in 5-alpha GFP expression between LB media and SOC media over 24 hours (Mann-Whitney, U=854, n=25,25, p<0.001). The GFP expression of the 5-alpha strains grown in both LB and SOC media decreased during the initial 4 hours of incubation, during the lag phase of growth followed by a substantial increase in expression in LB media over the remaining 20 hours and relatively constant expression level for the final 20 hours in SOC media.

2.2. Temperature:
The GFP expression of 5-alpha grown at 31ᵒC in SOC media at a neutral pH was 1.68x105 AFU/OD600 with the standard error of 6.10x103 AFU/OD600 after 24 hours of incubation. This was higher than the GFP expression in the 5-alpha grown at 37ᵒC and 43ᵒC, which were 4.08x104 AFU/OD600 with the standard error of 7.13x103 AFU/OD600 and 3.94x104 AFU/OD600 with the standard error of 4.38x103 respectively. There was a significant difference in mean GFP expressions of 5-alpha between 31ᵒC, 37ᵒC and 43ᵒC in SOC media over the 24 hours (ANOVA, F2,72=12.10, p<0.001). A post hoc Tukey test indicated that there was no significant difference between the expression at 37ᵒC and 43ᵒC (p>0.05) but the expression and 31ᵒC was significantly different to the other temperatures (p<0.001). The Gibson Assembly construct in 5-alpha also had a higher GFP expression in LB media compared to SOC media during the stationary growth phase at 31ᵒC.

2.3. pH:
The 5-alpha E.coli containing the Gibson Assembly construct which was grown in 37ᵒC SOC media at pH 9 had a higher final GFP expression than the 5-alpha grown at pH 7 or pH 5; final the GFP expression of 5-alpha at pH 9 was 4.12x105 AFU/OD600 with the standard error of, 2.40x104 whereas the GFP expressions for 5-alpha grown at pH 7 and pH 5 were 4.08x104 AFU/OD600 with the standard error of 7.32x103 AFU/OD600 and 2.88x104 AFU/OD600 with the standard error of 4.27x103 AFU/OD600 respectively. There was a significant difference in mean GFP expression levels between 5-alpha grown at pH 5, 7 and 9 over the 24 hours (ANOVA, F2,72=49.88, p<0.001). A post hoc Tukey test indicated that there was no significant difference in the 5-alpha GFP expression between pH 5 and 7 (p>0.05) but 5-alpha at pH 9 had a significantly higher GFP expression than at the lower pHs (p<0.05).
Additionally, the 5-alpha E. coli grown in 37ᵒC LB media had a higher GFP expression at pH 9 than the 5-alpha grown in pH 5 and pH 7 LB media; the GFP expression at pH 9 was 9.13x105 AFU/OD600 with the standard error of 3.45x104 AFU/OD600 whereas the GFP expression at pH 5 and 7 in LB media were 6.38x105 AFU/OD600 with the standard error of 2.49x104 AFU/OD600 and 6.18x105 AFU/OD600 with the standard error of 1.68x104 AFU/OD600 respectively.

2.4. Chassis Strain:
The BL21 E. coli strain containing the Gibson Assembly construct had a significantly higher GFP expression level than the 5-alpha and TOP10 strains when incubated in SOC media at 37ᵒC at a neutral pH; the BL21 GFP expression was 1.01x105 AFU/OD600 with the standard error of 3.66x104 AFU/OD600 at 24 hours whereas 5-alpha was 4.08x104 AFU/OD600 with the standard error of 7.32x103 AFU/OD600 and TOP10 was 4.04x104 AFU/OD600 with the standard error of 1.24x104 AFU/OD600 at 24 hours. There was a significant difference in mean GFP expression levels between 5-alpha, BL21 and TOP10 grown at 37ᵒC at a neutral pH in SOC media over the 24 hours (ANOVA, F2,72=15.16, p<0.001). A post hoc Tukey test indicated that there was no significant difference in the GFP expression between 5-alpha and TOP10 (p>0.05) but BL21 had a significantly higher GFP expression than the other strains (p<0.05).
TOP10 E. coli grown at 43ᵒC, pH 7 in LB had the highest overall level of GFP expression at 1.31x106 AFU/OD600 with the standard error of 8.65x104 AFU/OD600 which was during the stationary phase of growth at 20 hours. The second highest overall level of GFP was expressed by BL21 at pH 9 in 37ᵒC SOC media at 1.27x106 AFU/OD600 with the standard error of 7.24x104 AFU/OD600.

Internal Controls

Text goes here.

Robust Promoter Characterisation

Text goes here.

Conclusions and Future Work

Text goes here.

References

Text goes here.