Part:BBa_I746907:Experience

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User Reviews

TU_Munich Team 2011

We tested the part and it worked very well.

Contribution: Ulaval 2019

This parts was characterized by Team ULaval in the cell-free expression system myTX-TL from ArborBioscience. This part had never been characterized, and no fluorescence or expression data was available. By expressing this part in a cell-free commercial media, and measuring the intensity of GFP fluorescence, we hope to demonstrate that this part is indeed functional, and usable inside a cell-free expression system. As shown by figure 1, fluorescence was observed after 75 minutes, attained significant levels after about 5h and peaked after 16h.

Our data shows that this part is compatible with the cell-free expression system used. 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.

<img src="https://2019.igem.org/wiki/images/0/04/T--Ulaval--ResultsFig7B.png(79 kB) " style="width:900px;"> Figure 1. Fluorescence curve for BBa_I746907. 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.

TU_Munich Team 2011 GFP Assay

I746907 (T7 promotor with GFP) in pSB1A2 in BL21DE3 cells

change of medium to M63 (LB shows slight fluorescence itself)

five concentrations of IPTG in the medium were tested (1.5, 1.0, 0.5, 0.25, 0.1 mM IPTG) growth of cells at 37 °C, shaking

excitation filter: 485 nm, emission filter: 520 nm

Problem: measurement of Absorbance only every 100 minutes led to spikes in the diagram (Fluorescence measured every 10 min.)

results

part works well

induction t(1/2) = ca. 200 min

no concentration dependency of GFP induction at 1.5 to 0.1 mM IPTG, as IPTG concentration was in saturation range

for this diagram:

• calculation: Fluorescence/Absorbance
• mean of triplicate measurement
• adjusted to 100 %
• corrected for outliers
• shortened to 700 min

UNIQ0d7ecb8574c43f40-partinfo-00000000-QINU UNIQ0d7ecb8574c43f40-partinfo-00000001-QINU