Difference between revisions of "Part:BBa K2042004"

Latest revision as of 00:12, 30 October 2016

IPTG inducible promoter

This is the sequence of an leaky IPTG inductible promoter working in Pseudomonas putida, extracted from a SEVA (Standard European Vector Architecture) plasmid. [1]

The expression system was formatted following the SEVA standard as part of the reference plasmid collection. This consists of plasmids containing the IPTG-inducible lacIq-Ptrc system, which was excised from the pTrc99a vector and assembled in the SEVA frame as a PacI-AvrII fragment so that the regulated promoter points toward the AvrII site. In this way, users can benefit from the remaining downstream cloning sites of the default polylinker. Note also that this expression cargo (as well as any other SEVA counterpart) lacks translation signals (e.g. Shine-Dargarno ribosome binding sequences), which may need to be engineered on a case-by-case basis. [1]

In our project, we aimed to use this leaky promoter to induce the expression of genes in BBa_K2042013 and BBa_K2042014 for PLA production.

Information on IPTG

Isopropyl β-D-1-thiogalactopyranoside (IPTG) is a molecular biology reagent. The compound is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon, and it is used to induce recombinant protein expression where the gene is under the control of the lac operator. Like allolactose, IPTG binds to the lac repressor and releases the tetrameric repressor from the lac operator in an allosteric manner, thereby allowing the transcription of genes in the lac operon. But unlike allolactose, the sulfur (S) atom creates a chemical bond which is non-hydrolyzable by the cell, preventing the cell from metabolizing or degrading the inducer. [2]

References

[1] Silva-Rocha, E. et al. (2013) The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res. 41, D666–D675.

[2] Hansen, L.H., Knudsen, S., Sørensen, S.J. (1998). The effect of the lacY gene on the induction of IPTG inducible promoters, studied in Escherichia coli and Pseudomonas fluorescens. Curr. Microbiol. 36 (6): 341–7.

Characterization

This promoter was characterized by Fluorescent measurement. GFP was inserted under the control of the IPTG inducible promoter and transformed in Pseudomonas putida. Different experiments were performed.

For this experiment the cell grew in the 30ºC chamber with shaking until they reached OD of around 0.4 (spechtophotometre indicated that the OD was 0.4, the TECAN in which the fluorescence was read says 0.2 in the beginning, but this OD is irrelevant since the values only matter for normalization). When the OD was reached, induction with the indicated concentrations of IPTG was made, the culture was put into a 96-wells plate, and measured on the TECAN for 1hour. (Note, the measure was made without shaking) These are the results that the TECAN automatically provides.

For this experiment the cell grew in the 30ºC chamber with shaking until they reached OD of around 0.4 (spechtophotometre indicated that the OD was 0.4, the TECAN in which the fluorescence was read says it is 0.2 in the beginning, but this OD is irrelevant since the values only matter for normalization). When the OD was reached, induction with the indicated concentrations of IPTG was made, the culture was putted back in the 30'C chamber with shaking. Timepoints was taken and measured directly in the TECAN. These are the results. Values for graph without correction of OD600

Values for graph normalizing with OD600

5h Long washed with water The experiment was repeated because LB emits fluorescence at the same range as GFP. This time it was done like this: For this experiment the cell grows in the 30ºC chamber with shaking until they reached OD of around 0.4 (spechtophotometre indicated that the OD was 0.4, the TECAN in which the fluorescence was read says 0.2 in the beginning, but this OD is irrelevant since the values only matter for normalisation). When the OD was reached, induction with the indicated concentrations of IPTG was made and the culture was putted back in the 30'C chamber with shaking. Timepoints was taken, washed them with water and measured them directly in the TECAN IN WATER These are the results.

Values for graph normalizing: