Measurement Award

BioBricks used: BBa_J364001, BBa_J364004, BBa_J364005

Rationale and Aim

Text goes here.

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.

Interlab Devices in Different Contexts

We looked at the impact of temperature, pH and media on how high, medium and low expressing devices performed. Tests 2, 5 and 6 were selected from the Interlab Study after initial results were analysed. Single colonies of each transformant were selected and grown in LB+Chl, then washed and diluted to an OD600 of 0.05 in 100 ul media. Transformants were analysed in quadruplicate, with bacteria and media pipetted onto the plate using a pipetting robot for maximum accuracy. Once set up, the plate was incubated at specified temperatures in a plate reader with double orbital shaking, taking readings every 10 min for 24 h.

Temperature

Growth in Test 2 was affected differently in LB and SOC (Fig 3). In LB, there appears to be a slight increase in max OD reached as temperature increases, however at an alkaline pH the device appears unable to grow well in higher temperatures. In SOC, there is no distinct pattern in how temperature affects growth, however it is clear that the bacteria grows better as pH is increased, suggesting pH and temperature have a combined effect.

Figure 3 Max OD reached by Test 2 in (A) LB and (B) SOC media over 24 h at 31C, 37C and 43C.

Fluorescence levels were affected differently at different pH levels as temperature was increased (Figure 4). 37C appears to be optimum temperature for both media, as there are decreases between 37C and 43C at all pH levels apart from pH 7.20 in LB media. However, we can see a dramatic increase in overall max fluorescence levels in both LB and SOC when pH is increased to an alkaline level.

Figure 4 Max fluorescence reached by Test 2 in (A) LB and (B) SOC media over 24 h at 31C, 37C and 43C.

For the overall FL:OD, in LB media 37C was the optimum temperature, but in SOC a higher temperature appeared to be more favourable. In both media, the exception was seen at an alkaline pH, where FL:OD increases with temperature in LB, and 37C was optimum in SOC (Figure 5).

Figure 5 Max FL:OD reached by Test 2 in (A) LB and (B) SOC media over 24 h at 31C, 37C and 43C.

pH

Growth was affected significantly by changes in pH, which can be seen in figure 6. In SOC media, a distinct increase is seen in max OD as pH increases. In LB media the opposite is seen, where as pH increases, max growth decreases.

Figure 6 Max OD reached by Test 2 over 24 h in (A) LB and (B) SOC media adjusted to varying pH levels.

Fluorescence was affected by pH particularly in SOC where an increase was seen with an increase in pH; In LB there was less of a pattern seen; increases can be seen at 31C and 37C with increases in pH, however at 43C the max FL is reduced dramatically. The combination of high temperature/pH may have been toxic to the cells.

Figure 7 Max FL reached by Test 2 over 24 h in (A) LB and (B) SOC media adjusted to varying pH levels

Despite clear correlations in both growth and fluorescence, there appears to be no distinct pattern in the increase of pH and the overall FL:OD (Figure 8).

Figure 8 Max FL:OD reached by Test 2 over 24 h in (A) LB and (B) SOC media adjusted to varying pH levels

Overall, it is clear pH and temperature have an impact on the growth and fluorescence of the devices, and this raises the importance of maintaining conditions specified in InterLab instructions. It is difficult to draw definite conclusions without more thorough experimentation. It should also be noted that whilst each replicate of isolates was added to the plate using a robot for maximum accuracy, there was still a degree of variability. These variances will have increased over time, and the InterLab method of taking samples at specific time points may be more laborious, but will yield a more accurate result of overall culture behaviour.

Standard Assembly Methods

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).

Standard Measurement Methods

Text goes here.

Internal Controls

Text goes here.

Robust Promoter Characterisation

Text goes here.

Conclusions and Future Work

Text goes here.

References

Text goes here.