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.

Verify the expression of CAHS protein

Fig.2 the circuit of BBa_K2623014

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 the E.coli DH5а with BBa_K2623014 and IPTG turns red.(D+ is E.coli DH5а with BBa_K2623014 and IPTG, D- is E.coli DH5а with BBa_K2623014 without IPTG, D0 is E.coli DH5а without BBa_K2623014)

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

The results showed that CAHS protein could improve the preservation effect of freeze-drying bacteria significantly!
The B+ group’s CFU is 100 times higher than the B0 group, 30 times better than the B- group. So we think the CAHS is leaking expression in B- group, which has some of the protection for the bacteria in freeze-drying.
After being irradiated by UV for 1min, the B0 group was nearly fully dead, and the CFU of B- group had been decrease to a very low level, while the B+ group was higher. So we think the CAHS is can provide some of certain protection against UV.

Figure 6 The CFU of BL21. (B0 is E.coli-BL21 without BBa_K2623014, B+ is E.coli-BL21 with BBa_K2623014 and IPTG, B- is E.coli-BL21 with BBa_K2623014, UV means the desiccant bacterial under UV for 1min.)

This result shows that the CAHS can provide production for bacteria in freeze-drying. And the CAHS can also provide CAHS for bacteria suffering from UV. We think the CAHS protein is a very potential protective agent, so we can use the CAHS protein to store plasmid and bacteria. We can even design a kit for the bacteria storage with CAHS protein.
After being irradiated by UV for 1min, the B+ group was still alive. Therefore, we think the CAHS protein can provide protection for bacteria against UV and can protect the nucleic acid in the bacteria. The water bear can survive from UV, and the bacteria can also survive from UV, so it is certain the CAHS protein works.
As for the colony recovery rate, the CAHS also showed great effect. The B+ group colony recovery rate was highest, which reached about 40%. While those of the B- group and B0 group were no more than 2%. After being irradiated by UV for 1min, the B+ group was still the highest in the three group (UB_B0, UV_B+ and UV_B-).

Figure 7the colony recovery rate. (B0 is E.coli-BL21 without BBa_K2623014, B+ is E.coli-BL21 with BBa_K2623014 and IPTG, B- is E.coli-BL21 with BBa_K2623014, UV means the desiccant bacterial under UV for 1min.)

Here are the pictures of the culture plate. We used the disposable plastic petri dishes, and appended the sealing film on the aperture. When we started, we used the glass petri dishes, but the effect was not obvious at all. All the petri dishes had chloramphenicol, and the B0 group bacteria had transformed BBa_S0100 (pSB1C3 skeleton).

Figure 8: the plate of colony for counting.(B0 is E.coli-BL21 without BBa_K2623014, B+ is E.coli-BL21 with BBa_K2623014 and IPTG, B- is E.coli-BL21 with BBa_K2623014, UV means the desiccant bacterial under UV for 1min. And the 104& 105 means Dilution ratio)

Concluson

Our experiment shows the great function of CAHS protein in improving the desiccation tolerance and UV tolerance. We think that the CAHS protein is a very potential protective agent for bacteria storage. Except expressing the CAHS protein with plasmid, we can also knock the CAHS protein gene into the genome of bacteria through homologous recombination. And we believe that a new way for storing the bacteria is available.
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]