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From 2010.igem.org

We ran a number of experiments over the course of the summer that fell outside the usual. Given below are the most important of them.

Gradient PCR to troubleshoot NICE PCR extraction

Planning:

This experiment was run to troubleshoot a problem in the amplification of NICE (NISIN inducible expression system) promoter from pNZ8048. The amplification of the template was resulting in non specific bands most likely due to improper annealing of the primer to the template.
To test this a gradient pcr was run with a temperature range of 60-70˚C (Tm of primers = 68˚C)

Results:

No amplification was seen under any of the conditions, indicating that there is a high chance that the primers were designed incorrectly, providing inadequate annealing or that there was a large extent of inter-primer annealing at all temperatures.

Luciferase Assay for Acid Tolerance Promoter Charactrisation

Planning:

The experiment was run to study the expression of the p170 promoter using the firefly luciferase present in the pGL3 basic vector as a reporter. J23119 was used as a constitutive promoter to provide an expression plot in acid tolerance to aid in interpretation. We initially cultured colonies as primary inoculum till they reached an OD600 of 1. These cultures were then used to inoculate pH adjusted media maintained at 5 different pHs ( 4.5, 5, 5.5, 6, 7). We allowed these cultures to reach an OD600 of around 0.5 (to achieve a standard cell number) and then took samples of 1ml each.
To maintain statistical accuracy each run was done with 2 independent cultures and with each sample in triplicate. The readings were subsequently tabulated, averaged and normalized with Optical density of the culture sample.
Each sample was grown until stationary phase was achieved, to account for the growth phase induction of the P170 acid tolerance promoter.

Results:

The luminescence of the samples was measured in a arbitrary Relative Luminescence units/sec, we normalised these readings with the OD600 values of the cultures at the time of harvesting to obtain a rough estimate of luminescence per cell. The following are the tabulated values.


Table 1: Luminescence values of the cultures containing K372001 in pGL3Basic at various pH, normalised with respect to OD600



Table 2: Luminescence values of the cultures containing J23119+B0034 in pGL3Basic at various pH, normalised with respect to OD600
When plotted, it was seen that in the low pH range, K372001 duplicates showed opposing trends indicating a large amount of noise in our measurement. It is also noticed that from 5.5 - 6.0, there is a common increasing trend in both the cultures indicating that our experiment might be partially successful at capturing the dynamics of the P170 promoter.


Figure 1: Plot of K372001 activity at various pH in pGL3Basic with Luciferase as a reporter.

Most interestingly, it was seen that our constitutive reference promoter, J23119 with the RBS, B0034 showed extremely upregulated activity at the low pH range 4.5 - 5.5. This is probably due to another acid tolerance response that affects the constitutive promoter.


Figure 2: Plot of J23119+B0034 activity at various pH in pGL3Basic with Luciferase as a reporter.

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