Composite
CAHS 1

Part:BBa_K2623014

Designed by: Hongling Liu   Group: iGEM18_XMU-China   (2018-09-28)
Revision as of 12:58, 17 October 2018 by Bb1984 (Talk | contribs) (→‎Verify the role of CAHS protein in preserving biological activity)


Cytosolic-abundant heat soluble protein "CAHS " (promoter, RBS, RFP, lacI and double terminator)

Summary

This part contains the coding region of the Hypsibius dujardini (Water bear) (Macrobiotus dujardini) CAHS gene. Cytosolic abundant heat soluble proteins acting as a molecular shield in water-deficient condition. Tardigrade-specific intrinsically disordered proteins (TDPs) are essential for desiccation tolerance by forming non-crystalline amorphous solids upon desiccation, and this vitrified state mirrors their protective capabilities.
CAHS is a cytoplasmic-abundant protein which can strength the bacteria stress tolerance under the conditions such as dehydration. So taking biosafety into account, we add a constant promoter BBa_J23100 into the CAHS circuit to express Lac I BBa_S0100 in order to inhabit the initiation of the Lac O BBa_R0011 promoter which can only be activated and then expressing CAHS and mRFP BBa_J04650 after IPTG is added.
The separate fragment of CAHS is 714bp, and the whole circuit is 2934bp.The plasmid skeleton is pSB1C3.


CAHS_Fig1.png

Identification

When we are building this circuit, we are doing the nucleic acid gel electrophoresis experiment to verify. After the circuit is built, we send the plasmid to sequence, and get the correct sequencing.
After new molecular cloning experiments, we do Enzyme-Cut identification to certify the plasmid is correct. We use the EcoRI and PstI to cut the plasmid, then we getting two fragments - one is 2932bp, and the other is 2029bp(Fig.1).

Fig.1 The result of this plasmid cut with enzyme EcoRI and PstI.































Verify the expression of CAHS protein


Fig.2






In the circuit, the Lac I was expressed constantly, and Lac O, lac repressor was bound to Lac O. Following the CAHS gene was our reporter gene mRFP (J04650). When we added IPTG in the bacterial solution, the IPTG bound to repressor and inactivated it. So the CAHS gene and mRFP would start to express.

Fig.3













We foundthat the red fluorescent protein expressed very slowly. After we added IPTG in the 4mL bacterial solution, we put the bacterial solution in 25° shaker culture. After 12 hours, we saw the centrifugal precipitation color showed red.<br. We also did experiment with E.coli BL21, but the bacteria didn’t turn red.

We cultured the E.coli BL21(DE3) which contained the plasmid of CAHS protein (BBa_K2623014) in the fluid medium. When the value of OD600 reached about 0.6, we took 4ml bacteria solution in the glass test tubes and added 4μL IPTG in the tubes. And then put them in 25° shaker culture for 5hs. Then, we took 1mL bacterial liquid to centrifugalize. We picked the precipitation and added 100μL DDW and 20μL SDS loading buffer.After that, we heated and boiled it for 15 minutes. And then, it was separated by SDS-PAGE and stained with coomassie brilliant blue for 40 minutes. Ultimately, we decolorized it and observed the results.
We find the CAHS is surely expressed in the E.coli BL21 after adding IPTG. Here is the result.
We have done the Protein expression Identification. According to the figures, we can see significantly increased protein content in the bacteria containing the plasmid. Here are the results(Fig.4) :

Figure 4: the CAHS protein express in SDS-PAGE.(1, 2, 3 is all with BBa_K2623014. 1 is BL21 with IPTG and pre-heat, 2 is BL21 with IPTG, 3 is BL21 without IPTG, 4 is BL21 without the BBa_K2623014.)






























The picture of SDS-PAGE shows the CAHS protein is surely expressed in B+ group (the E.coli BL21 with BBa_K2623014 with IPTG). And CAHS protein isn’t expressed in the B- group (the E.coli BL21 with BBa_K2623014 without IPTG) and B0 group (the E.coli BL21 without BBa_K2623014).
We also did experiments with E.coli DH5а, but the SDS-PAGE showed there was no CAHS protein. Because the E.coli BL21 is a kind of strain which can knock out proteases while the E.coli DH5а not. We guess the CAHS protein in E.coli DH5а is degraded. So we chose the BL21 to verify the function of CAHS.

Verify the role of CAHS protein in preserving biological activity

The SDS-PAGE showed the CAHS protein was really expressed in BL21. We cultured the E.coli BL21 bacteria which contained the plasmid BBa_K2623014. We picked the E.coli BL21 bacterial liquid for freeze-drying. First of all, we picked 1mL bacterial liquid to centrifugalize, added 100μL protective agent (LB+5% Sucrose), and put them in -20°refrigerator for 2h. And then, we put them in -80° for one thorough night, and then freeze-dried them (Advantage ES-53).
After lyophilization, we added 500μL PBS in each centrifuge tube, and put them in 37° shaker culture for 15mins. We also added 500μL DDW in each centrifuge tube. And then, we took 100μL the dilute bacteria liquid, added in 900μL DDW, repeated the step, and carried out gradient dilution to 104, 105, 106. Then, we picked 200μL dilute bacteria liquid, and coated the plate with chloramphenicol.

Figure 5 gradient dilution

For more information, please go to our result page.http://2018.igem.org/Team:XMU-China/Results











Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 375
    Illegal NheI site found at 1679
    Illegal NheI site found at 1702
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2880
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1376
    Illegal AgeI site found at 1488
  • 1000
    COMPATIBLE WITH RFC[1000]


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