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Part:BBa_K3736002
Plux promoter + RBS + LL37 + RBS + mRFP + RBS + Terminator*2
Plux promoter + RBS + LL-37 + RBS + mRFP + RBS + Terminator*2
Gene Construct of DenTeeth
We incorporate the whole part into E. coli BL21(DE3). We did colony PCR and digest to check its genotype.
mRFP Model
We inserted mRFP into our biobrick to detect the expression of the sterilization sequence in DenTeeth. The following graph was the simulated expression curve of mRFP.
LL-37 Model
Through the modeling built with substituting parameters from published articles, the growth of E. coli and P. gingivalis under the effect of LL-37 would be inhibited significantly. However, the figure also shows that E. coli could still live with LL-37. Simply speaking, we successfully test the feasibility of DenTeeth and find that we can improve the DenTeeth with both biobrick design and efficiency optimization model.
Because P. gingivalis was in the RG2, so we could not do the LL-37 functional test by inhibiting the growth of P. gingivalis. After reading some related papers, we found that the killing rate of E. coli was similar to that of P. gingivalis [4]. Based on these data, we determined to make E. coli, DH5α with pET32A, as the bacteria killed by DenTeeth in the LL-37 functional test.
LL-37 Functional Test
After finishing the design of DenTeeth, we wanted to know whether its inhibition ability can have a function, so we designed the following experiment.
By taking the method of dish culture and sticking up the filter paper, we dropped the DenTeeth on the filter paper in the center of the E. coli, DH5α with pET32a, plate. After twelve hours, a circular area around the spot of the DenTeeth formed, in which the bacteria colonies did not grow. Inhibition zone proved that the LL-37 produced by DenTeeth could successfully secrete out and inhibit the growth of E. coli, DH5α with pET32a.
![](/wiki/images/8/87/T--NCTU_Formosa--Zone_of_inhibition.png)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1650
Illegal NgoMIV site found at 1771
Illegal AgeI site found at 778
Illegal AgeI site found at 890 - 1000COMPATIBLE WITH RFC[1000]
None |