Part:BBa_K2250005
Pconst-RhlR-Prhl-LacI-PlacI-CmR
This is the reception part of warrior 2/beggar 2/farmer 2 in our project. This part makes warrior 2/beggar 2/farmer 2 able to recept the C4HSL secreted by warrior 1 so that the warrior 1 can kill warrior 2/beggar 2/farmer 2. This can be realized by C4HSL binding to RhlR, activating the expression of LacI, and then LacI inhibiting the expression of CmR, making the warrior 2/beggar 2/farmer 2 unable to survive under the Chloramphenicol.
I. How the part work
Fig.1 Gene circuit of BBa_K2250005
C4HSL (a kind of AHL) enters bacteria from the medium and then forms a complex with the protein RhlR. This complex activates the promoter Prhl, thus activating the expression of LacI. The expression of LacI inhibits the promoter Plac, thus inhibiting the expression of CmR. Therefore, when C4HSL is introduced, bacteria cannot grow well in medium containing chloramphenicol.
In this part, it is commonly known that CmR can be inhibited by C4HSL since C4HSL-RhlR-Prhl are well-paired, as is shown in Fig.2.
Fig.2 Dynamic response of RhlR-Prhl to C4HSL In this figure, RFP intensity is used to indicate response intensity and C4HSL is produced by bacteria itself which also has RhlR and Prhl. We can see that RhlR-Prhl respond to C4HSL remarkably after some time.
Besides, we hope that this part has low crosstalk with 3OC6HSL, i.e. only C4HSL can activate expression but 3OC6HSL cannot.
II. Low crosstalk between 3OC6HSL and RhlR-Prhl
To test if this part satisfies if 3OC6HSL will inhibit the expression of CmR, we put this part on pSB6A1, and put 3OC6HSL synthase LuxI (BBa_K2250007) on pSB3K3. These two parts were co-transformed into E.coli MG1655 ΔsdiA ΔlacI. The E.coli were cultured in medium containing ampicillin + kanamycin and chloramphenicol respectively. If CmR is not inhibited by 3OC6HSL, then bacteria’s growth under two conditions will be similar and our experiment shows that it is true.
Fig.3 Bacteria mentioned above does not kill itself
The figure above shows the result. C represents the abundance of warriors in chloramphenicol medium and A+K means the abundance of warriors in ampicillin + kanamycin medium. We can see from the result, the number of warrior under these two conditions are similar, so 3OC6HSL cannot activate Prhl via RhlR to inhibit CmR, which work as we expected.
Detailed method can be seen at [http://2017.igem.org/Team:Tsinghua-A/killing_test Killing test---2017 Tsinghua-A]
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2159
Illegal BamHI site found at 301 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 776
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