Difference between revisions of "Part:BBa J23119:Experience"

Experience of the IIT_Madras iGEM 2010 team.

The iGEM 2010 IIT Madras team worked with J23119 as a reference in order to characterise a new pH inducible promoter that we have added to the registry this year. While analysing the results of the charactersation experiment we conducted we found that this promoter, in conjunction with an RBS (J23119 + B0034) has upregulated activity at low pH (4.5 - 5).

Experimental Design

The experiment we conducted involved the use of the construct J23119+B0034 in the plasmid pGL3Basic, which is a promoter-less plasmid that contains Firefly Luciferase as a reporter. The plasmid was transformed into DH5Alpha and cultured as a primary inoculum in Luria Bertani media. Once the inoculum reached an OD600 of 1.0 the culture was used to inoculate pH adjusted media of pH 4.5, 5, 5.5, 6 and 7, all in duplicates. We regularly measured OD600 for these 10 cultures until their OD600 reached 0.5, or reached saturation. We then took 3 independent samples of these cultures with vigorous shaking in-between consecutive samplings.

Observations and Inferences

In this way we obtained a measure of the Luminescence of 30 samples in an arbitrary unit, RLU/s (Relative Luminescence units per sec). The data is as tabulated below.



Table 1 and 2: Data representing experiment done at various pH in duplicate and sampling done in triplicate. The Luminescence has been normalised using harvest-time OD600 readings of each culture.

The data was also plotted, as shown below.



Figure 1: Experimental data plotted for the duplicates showing high level of reproducibility in the data.

From these results we infer that it is highly possible for the constitutive promoter J23119 to be under the control of another stress factor based on pH. In this case it is possible that due to the high pH stress in the medium, the luciferase is over-expressed. It should also be noted that our pH inducible (in the range of 5 - 6.5) did not show this increased activity at low pH.

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References

• pGL3Basic vector backbone, as distributed by Promega. http://www.ncbi.nlm.nih.gov/nuccore/13195703, last accessed on 30th Oct 2010, 0350 IST (GMT +0530).
• DH5Alpha E.coli strain. http://ecoliwiki.net/colipedia/index.php/DH5_alpha, last accessed on 30th Oct 2010, 0350 IST (GMT +530).