Part:BBa_K2623014
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.
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).
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.2) :
Comments:
1. BL21 with this plasmid, IPTG+, and after Ultrasonic crushing and protein heat treatment.
2. BL21 with this plasmid, IPTG+.
3. BL21 with this plasmid, IPTG-.
4. BL21 with S0100(pSB1C3).
Verify the expression of CAHS protein
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.
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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.
For more information, please go to our result page.http://2018.igem.org/Team:XMU-China/Results
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 375
Illegal NheI site found at 1679
Illegal NheI site found at 1702 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2880
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1376
Illegal AgeI site found at 1488 - 1000COMPATIBLE WITH RFC[1000]
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