Difference between revisions of "Part:BBa K1824018"

Revision as of 15:50, 17 September 2015

aeBlue Blue chormoprotein with lva tag

AeBlue chromoprotein could be expressed as a deep blue color in E.coli, which was added by Uppsala University in 2012. We add lva tag to before stop codon to increase the degradation of the Aeblue chromoprotein, which is necessary in our earth map for color changing.

Characterization

XJTLU-CHINA tested accumulation status of blue chromoprotein with two different fast digestion tag,lva and aav tag, under the regulation of T7 promter-lac operator in E.coil strain BL21 (DE 3). The construction was shown in Figure 1.

Figure 1: The constructions that XJTLU-CHINA made to perform chromoprotein accumulation test.

Three groups were used in experiments: AeBlue chromoprotein, AeBlue chromoprotein with AAV tag and AeBlue chromoprotein with LVA tag. Two mono-colonies were chosen from each group. 300ml LB was used to culture the 6 colonies at 37 degrees Celsius for 4 hours. Then 300ul IPTG was added to induce at 16 degrees Celsius for 12 hours. The data was collected from 5 hours to 12 hours, totally 13 points. For each point, 20ml colony liquid was obtained from the 300ml LB culture. 4ml of them was used for OD measurement at 600nm and the rest 16ml of them was used for protein extraction.

Firstly, 16ml bacteria were centrifuged at 6000 rpm for 5 minutes. Then the supernatant was discarded and 10ml PBS was added to resuspend the sediment. After using ultrasonic wave to break the cell, 4ml of mixture was centrifuged at 12000 rpm for 5 minutes. Finally, the absorbance of blue chromoprotein was measured at 597nm by spectrophotometer.

Results as shown in diagram below.

Sequence and Features