Difference between revisions of "Team:AHUT China/New Parts"

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<p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 16px;">&nbsp;&nbsp;&nbsp;Fig. 1-1 Map of CA2-WT recombinant vector</p>
 
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Revision as of 06:06, 12 October 2018

Royal Hotel Royal Hotel

Experimental results




    BBa_K2547000: Construction of CA2-WT expression plasmid

     We first synthesized the sequence of human carbonic anhydrase CA2, and then cloned it into the expression vector pET-30a(+), and identified the correctness of the obtained recombinant vector by restriction enzyme digestion and sequencing (Fig. 1-1 and Fig. 1-2).




   Fig. 1-1 Map of CA2-WT recombinant vector

8.30

    Pilot Expression of Protein
   “IPTG+ 4h”“IPTG- 4h”

 

 


   After the rocker culture is 4 H, add IPTG(final concentration is 0.5 mm) and then the rocker culture is 4 hours. Each of the two tubes absorbs 200 μL of bacterial fluid, and in the frozen centrifuge, it is 4 °C and 12000 rpm centrifuge. 10 minutes, go to the clear, It is resuspended with 100 μL 1x SDS Loading buffer, then heated in a water bath pot at 100 °C for 10 minutes and stored in a -20 °C refrigerator.

    8.31

    Protein extraction
    CA2L203K

 

 

    CA2WT
    CA2L203K




    9.01

    Purification of Protein
     Prior to purification


    Purified





   9.03

    Western blot identification of proteins
     CA2L203K    CA2WT





   9.04

    Quantification of protein concentrations





   9.05

    Colorimetric enzyme activity
     Add 250 μl saturated CO2 solution


   According to the average calculated in the table above, the unit enzyme activity of the mutated carbonic anhydride enzyme is 0.3710(10 5 WAU/μg), and the unit enzyme activity of wild carbonic anhydride enzyme is 0.1755(10 5 WAU/μg).
Test esterase activity
Determination curve without carbonated anhydrase:



   Determination curve with carbonated anhydrase（CA2WT）


   Determination curve with carbonated anhydrase（CA2L203K）


   Wild carbonic anhydrase activity calculated as 0.5098 mol/g × min
   The mutated carbonic anhydrase enzyme activity was calculated to be 1.0392 mol/g * min.