Difference between revisions of "Part:BBa K3187011"

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Name	pTeTW3con2-ptet-mCherry--sfGFP-pT7
Base pairs	1780
Molecular weight	26.7 kDa (mCherry) + 26.8 kDa (sfGFP)
Origin	Synthetic
Parts	tetA promoter, T7 promoter, T7 terminator, Lac Operator, RBS, mCherry, sfGFP
Properties	Dual expression of mCherry and sfGFP as reporters to monitor expression levels of the tetA and T7 site.

Usage and Biology

This composite part is a dual expression plasmid with pTeTW3con2 as backbone. It was cloned to characterize the expression levels of the two convergent expression sites. The tetA promoter is inducible with anhydrotetracycline (AHT) and the site (BBa_K3187039) encodes the protein mCherry. The T7 promoter can be induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and the site (BBa_K3187040) encodes the protein sfGFP.

The fluorescence of mCherry and sfGFP can be measured and acts as expression reporters.

Results

Cloning

pTeTW3con2-ptet-mCherry--sfGFP-pT7 was cloned in two steps via a restriction and ligation protocol. First, the mCherry gene was cloned into the backbone pTeTW3con2. Sequencing analysis was carried out to test whether the cloning was positive before the next step started. Next, the sfGFP gene was cloned into the backbone (pTeTw3con2-ptet-mCherry). The cloning fo the final product was checked via sequencing.

Measuring the expression levels after single induction

The measurement (fig. 1 and 2) showed a strong background expression of the T7 site represented by the increasing fluorescence signal of sfGFP. However, this background expression of sfGFP lessend with a rising AHT concentration. This came as quite the surprise, since inducing with different AHT concentrations was supposed to mainly regulate the tetA regulated site. Generally, the data shows a clear excess of the sfGFP fluorescence.

Unfortunately, we were not able to select the fitting settings for monitoring the fluorescence signals of sfGFP and mCherry. The final fluorescence signal of sfGFP in IPTG induced triplicates was stronger than the maximal detection point of the instrument while a change in the signal of mCherry was barely detected during the measuring process. For this reason, we changed our method to an expression assay where the fluorescence was detected after a semi denaturized SDS PAGE via a fluorescence imager.

Testing various dual induction strategies

Tuning the expression ratio