Measurement Award

BioBricks used: BBa_J364001, BBa_J364004, BBa_J364005

Rationale and Aim

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Background Information

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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.

pH

Growth was affected significantly by changes in pH, which can be seen in figure 3. 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).

Figure 5 Max OD reached by (A) Test 6, (B) Test 5 and (C) Test 2 in SOC media over 24 h at 31C, 37C and 43C.

Figure 6 Max OD reached by Test 5 in LB media over 24 h

Fluorescence was affected by pH particularly in SOC where an increase was seen with an increase in pH; In LB there was little change in fluorescence until an alkaline pH was reached at 43C, where there was a substantial decrease (figure 7).

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

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

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Standard Measurement Methods

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Internal Controls

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Robust Promoter Characterisation

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Conclusions and Future Work

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References

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