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).

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

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) :

Fig.2 the expression of CAHS in E.coli BL21

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

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. 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]