Difference between revisions of "Part:BBa K1033903"

Revision as of 19:56, 19 October 2021

tsPurple, purple chromoprotein (incl RBS, J23110)

This chromoprotein (also known as TinselPurple) naturally exhibits strong color when expressed.

Usage and Biology

This part is useful as a reporter.

iGEM2013 Uppsala: The images above show E coli constitutively expressing the chromoproteins amilCP BBa_K592009 and tsPurple BBa_K1033906 from the high copy plasmid pSB1C3 from the promoters J23116 and J23110.

Contribution

Group: Linkoping_Sweden iGEM 2021
Author: Ewelina Bladh
Summary: In this contribution, we recorded color development in colonies of E.coli cells of strain XL1. In addition to studying the color development, we determined tsPurple's absorbance maximum wavelength.

Color Development
In order to determine the progression of color development in colonies, E.coli cells (XL1) were plated onto LB-Miller agar plates containing a concentration of 25 ug/mL chloramphenicol. The cells were plated in two ways after transformation. The first by plating 100 µL immediately from the liquid culture. The second by first centrifuging the culture, discarding 90% of the supernatant and resuspending the cell pellet in the remaining 10%, followed by plating 100 µL of the resuspension. This results in what is from now on referred to as the diluted and concentrated plate, respectively.

These plates were incubated in 37°C for the following four days, where they were checked upon each day. Figure 1, displayed below, shows the diluted and concentrated plates of tsPurple during this four day study. The illustration of the diluted plate shows that after 12 hours, no color can be seen to the naked eye. This changes over the course of the next 24 hours, where at 36 hours the colonies on the concentrated plate are clearly visible. The diluted plate appears to need more time to properly express tsPurple, and colonies are not clearly visible until 60 hours after plating. The color intensity continually increases after the first notion of color in the colonies. Additionally, the concentrated plate shows indications of colored colonies already at 12 hours.

Absorbance and Fluorescence
Single colonies displaying a purple color were picked from the diluted plates to create liquid cultures. The liquid cultures consist of 5 mL LB-Miller media and a concentration of 25 µg/mL chloramphenicol. The liquid cultures were incubated at 37°C and 200 rpm for the next two days to ensure protein expression. After incubation, the liquid cultures were centrifuged until a pellet formed. The supernatant was discarded, and the remaining pellet was placed in a -20°C freezer. Cell lysis was done using both water and sonication. The frozen pellet was thawed and 5 mL autoclaved dH₂O was added, followed by vortex treatment to resuspend the pellet. Sonication on ice was the next step taken to lyse any cells that did not lyse upon the addition of water. The amplitude of the sonicator was set to 30%, 30 seconds pulsing followed by 30 seconds rest for a total of 2 minutes. Following sonication, the cultures were centrifuged until a pellet formed whereupon the supernatant was transferred to a clean 15 mL Falcon tube and the pellet was discarded. The supernatant of tsPurple can be see to the left in Figure 2. Simultaneous experiments with other chromoproteins was also made, their supernatant can be seen with tsPurple to the right in Figure 2. The following chromoproteins were also assessed in the same way as tsPurple: meffBlue (BBa_K1033900), amajLime (BBa_K1033914), spisPink (BBa_K1033923), gfasPurple (BBa_K1033917), and asPink (BBa_K1033926).

As a control, plates were also made with untransformed XL1-cells. A colony was picked from the diluted plate to start a liquid culture, followed by lysis like the cells transformed with tsPurple. Treatment of control and experimental cells was identical apart from the control culture not being exposed to antibiotics. The supernatant of the control can be seen to the right in Figure 2 along with other chromoproteins.

In order to measure the absorbance, 100 µL supernatant of tsPurple and 100 µL supernatant of the control were pipetted into a 96-well plate and measurements were made with BMG Clariostar plate reader. Measurements of absorbance were taken every 2 nm in the range 300 nm to 850 nm. This interval was chosen to include the entire visual spectra including margins. The data yielded from the measurements were subsequently baselined by subtracting the control's absorbance values from tsPurple's absorbance values followed by normalization. The resulting graph for tsPurple's absorbance can be seen in Figure 3 below. The absorbance maximum of tsPurple proved to occur at approximately 580 nm.

When examining the supernatant extracted from tsPurple-expressing cells on a UV-table, no fluorescence was detected. The chromoprotein amajLime (BBa_K1033914) was studied at the same time, functioning as a positive control, and the lysate from the control functioned as a negative control.

Sequence and Features