Team:HIT-Harbin/Experiments

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HIT-Harbin

Project/experiments

Experiment Schedule

JuL. 10~15 Preparation of media, competent cells and experimental reagents

JuL. 16~20 Preparation of parts from iGEM

JuL. 21~31 Respective ligation of strong, intermediate and weak RBS with sub-circuit hrpR/hrpS/tet/RFP

Aug. 1~10 Ligation of terminators

Aug. 11~20 Successful ligation of the four sub-circuits

Aug. 21~28 Combination of sub-circuits: the device

Aug. 29~Sep. 15 Test of the device

Sep. 16~25 Remaining experiments

Fig 1. PCR resuLts of our own parts

(1: hrpL; 2: hrpS; 3: hrpR; 4: Ptet +strong RBS+hrpS+T; 5:Ptet+intermediate RBS+hrpS+T; 6: Ptet+weak RBS+hrpS+T; 7: PIPTG +strong RBS+hrpR+T; 8 is not needed; 9: PIPTG +weak RBS+hrpR+T; 10: PhrpL+strong RBS+tetR+T; 11: PhrpL+intermediate RBS+tetR+T; 12: PhrpL+weak RBS+tetR+T; 13: PhrpL+strong RBS+RFP+T; 14: PhrpL+weak RBS+RFP+T; 15: PhrpL+weak RBS+RFP+T)

Fig 1.EcoR1 and Pst1 double restriction enzyme cleavage for hrpL AND gate(BBa_K1014014) device and B-POM1(BBa_K1014999)

1:plasmid carrying BBa_K1014014; 2:double restriction enzyme cleavage for 1; 3:plasmid carrying BBa_K1014999; 4:double restriction enzyme cleavage for 3

1.Test of device

1)Preparation of IPTG solution: add 240mg IPTG powder into 10mL dd H2O. We filtrated the solution to sterilize it and broke it into EP tubes. The concentration is 24mg/mL (100mM/mL). then we stored them in -20℃.

2)To test the four different combination according to the strength of promoters, we made different concentrations of IPTG for the bacteria.

Table 1 IPTG formula

We grew the IPTG-added culture in 37℃ at 120rpm, overnight. Unfortunately, we haven’t observed the expected color of RFP (Red Fluorescence Protein).

Fig 3. No red after night

3)After that, we tested the reporting sub-circuit of our device. We connect the constitutive promoter PLac with RBS+RFP+T, transformed the bacteria and grew it overnight. The red in the results proved that the reporting sub-circuit is working.

Fig 4. Constitutive promoter expressing RFP

(Left is 12h, right is 24h)

4)We sent our whole device to companies for sequencing, but it failed.

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The circuit

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The circuit

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