Part:BBa_K567018:Experience

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Contribution: Ulaval 2019

This parts was characterized by Team ULaval in the cell-free expression system myTX-TL from ArborBioscience. The fluorescence intensity of this part had only been characterized in vivo, and no data was available for the use of this part outside of a cellular system. By expressing this part in a cell-free commercial media, and measuring the intensity of GFP fluorescence, we hope to provide other teams with data that shows that this part can be used outside of cellular systems. As shown by figure 3, fluorescence was observed after 75 minutes, attained significant levels after about 5h and peaked after 16h.

While this part was not designed with cell-free systems in mind, our data shows that it is indeed appropriate for use. Using this part in cell-free systems could ease the purification of this part if large quantities of the purified protein are necessary for a given experiment.

Figure 1. Fluorescence curve for BBa_K567018. Fluorescence intensity was measured and corrected according to the IGEM data analysis table v2. However, as these fluorescence measurements were carried outside of cells, no OD600 was calculated. Fluorescence is therefore only corrected using the fluorescein reference curve.

Message Marianne Côté

User Reviews

UNIQ3bafce2f503e533d-partinfo-00000001-QINU

•• kosyumote

The Pitt iGEM 2015 team (Group: iGEM15_Pitt (2015-09-18)) characterized this part in NiCo21(DE3) cells (a BL21(DE3) derivative). This part exhibits significant leaky expression due to the lac operator present in high copy number. For applications where leaky expression is deleterious, this part does not work. However, it produces easily quantifiable GFP which can be useful in many applications. Finally, we did not expect RFP fluorescence in NiCo21(DE3) or DH5a cells, since neither of them have amber suppressors, and none was observed.

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••• Shadowburn

Five pH gradients were set and fluorescence intensity was measured in five media. In each group of experiments, we repeated six times. The relative fluorescence intensity was obtained by dividing the average fluorescence intensity of the six groups by the optical density. We take the relative fluorescence intensity as dependent variable and the pH value as independent variable, and get the broken line diagrams of six points. Three different broken lines were obtained according to the different experimental time. It can be seen that the trend of relative fluorescence intensity is basically the same at different time points. The relative fluorescence intensity tends to decrease in the region with pH less than 6; it is basically stable in the region with pH 6-8; and it tends to increase in the region with pH greater than 8. In our opinion, the expression effect may be better in acidic or alkaline environment; another possibility is that our experimental measurements deviate in the environment of pH 6-8.

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•••• janniina

Because of the T7 RNA polymerase binding site we were not able to express the biobrick directly in our amberless host strain with pEVOL-pAzF plasmid, that expresses a tRNA synthetase able to add p-azido-L-phenylalanine in amber stop codons. When we used our improved this biobrick by taking the T7 binding site out, we were able to detect both GFP and RFP with a fluorometry. However, the RFP was not visually detectable, and since it is a fusion protein of the two, it is unlikely human eye would be able to separate the red and green from each other. It is more likely to look something else, like yellow, if the production is significant enough.

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