Coding

Part:BBa_K1824019

Designed by: Zhe Yang   Group: iGEM15_XJTLU-CHINA   (2015-09-14)

AeBlue blue chromoprotein with aav 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.


Aeblue.png
Aeblue lva.png
Aeblue aav.png
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.


Ud- variation of chromoprotein.png
Figure 2: The variation of chromoprotein after induced 300 mins.


Aeblue color.jpg
Figure 3: The color of non-tag, AAV tail and LVA tail after induced 720 minutes.


As shown in the Figure 2, both blue chromoprotein with LVA tail and AAV tail had less accumulation amount than the group of no-tail, which indicated both LVA tail and AAV tail raised the degradation rate. In addition, blue chromoprotein with LVA tail had an even lower accumulation in host cells compared to blue chromoprotein with AAV tail. It was speculated that the LVA tail provided a higher degradation rate. In conclusion, the tails can speed up the degradation of chromoprotein, and LVA tail is highly effective in protein degradation, whereas in our project the AAV tag was chosen to gain a balance between clear color performance and high speed of degradation.

References

Anderson,J. (1998) ‘New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria’, Applied and environmental microbiology, 64(6), June, pp.2240-2246.

Sequence and Features

Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 722
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 722
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 722
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 722
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal PstI site found at 722


[edit]
Categories
//cds/reporter/chromoprotein
//function/reporter/pigment
Parameters
None