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 in the uninduced condition. 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

The results of the samples which were collected after 6 h of expression (fig. 3) showed a distinct trend. Whenever IPTG was induced during the experimental procedure there was a large excess of sfGFP in comparison to mCherry. The induction times or used concentrations of IPTG just had a small effect on the resulting ratio of sfGFP to mCherry. Only the variant which was just induced with AHT showed a significant difference having a 2:1 ratio of sfGFP to mCherry. As expected from the previous experiments, the uninduced control showed the highest excess of sfGFP in this series.

The next samples were collected after continuing the expression overnight (fig. 4). These samples showed the same trend as the ones taken after 6 h of expression. As before, the difference of the various induction strategies in which IPTG was used showed no significant difference and the uninduced control had the largest excess of sfGFP in this series as well. Unlike the other samples, the AHT only induced variant showed a 1:1 ratio of sfGFP to mCherry.

Resulting, the data showed that the T7 site had under any induced condition much a higher activity than the tetA site. The expression was to strong for an effective tuning of the expression levels while IPTG was induced. Surprisingly, the background expression of the T7 site compensated the AHT inducted expression of the tetA site over night and was even stronger at 6 h after induction.

Tuning the expression ratio

The spectrophotometric measurements (fig. 5 and 6) of AHT induced trplicates showed a decline in the production of sfGFP with an increasing AHT concentration while the mCherry production seemed relatively constant at all tested concentration. These results were unexpected but provided us with a way to produce varying ratios of sfGFP to mCherry in dependence to the AHT concentration.

As can be seen in fig. 7, inducer concentrations of AHT ranging from 0.1 µg/mL to 0.4 µg/mL caused a change in the ratio of mCherry:sfGFP from 1:2 to 2:1 for overnight cultures of E. coli. The ratio seems to approach a maximum of around 2.2:1 following an increasing inducer concentration.

After the plotting of the collected data, we did an software-based regression by testing different types functions. The best fitting function with the highest determination coefficient was an asymetric sigmoidal function as presented in equation 1. The determination coefficent of this function is 0.989 which shows the great reliability of this function for further applications.

$$y = {0.4756 + {2.5966 \over (1 + 10^{((0.3047 - x) * 11.33)})^{0.2072}}}$$