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

Ligation for our device

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.

2.Investigating the relationship between the concentration of RFP with that of IPTG

1)Measuring absorbance of RFP

We grew bacteria without device and bacteria with our device in same volume until stationary phase. Taking bacteria without device as background, we measured the absorbance of bacteria with our device (the max absorption peak is 504nm).But absorbance in 504nm is higher than 1,which present a bad linear relation between absorbance and concentraton. RFP has absorption in 450nm,and absorbance is between 0.1 and 1(better linear relation),occasionally, we find a RFP standard curve under 450nm on the web. it was very lucky compared with our failure in test for our device. Before the mensuration, we diluted the two groups according to table2. We took the mean of two measures as the useful data.

Fig.5 RFP absorbance varying with wave length

Table 2 Dilution of Two groups of bacteria

Fig 6. The relationship between RFP concentration and absorbance(OD450)

2)The actual relationship between RFP concentration and absorbance

Fig 7. RFP standard curve obtain from the web,Click here

Through the standard curve, we can convert the relative concentration to the absolute concentration, and finally get the relationship between IPTG concentration and RFP concentration.

Compared to crushing cells to separate RFP, our method is simpler and easy to practice. Moreover, our relative concentration curve is credible. If the standard curve is reliable, our calculated result of RFP will be precise.