Team:HIT-Harbin/Experiments

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<h4 class="hashed"><span>The circuit</span></h4>
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<h4 class="hashed"><span>1.Test of device</span></h4>
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                             <p></p>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℃.
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<p></p>2)To test the four different combination according to the strength of promoters, we made different concentrations of IPTG for the bacteria.
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<p> Table 1 IPTG formula</p>
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<img src="https://static.igem.org/mediawiki/2013/a/a9/Experiment4.png"/>
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<p></p>We grew the IPTG-added culture in 37℃ at 120rpm, overnight. Unfortunately, we haven’t observed the expected color of RFP (Red Fluorescence Protein).
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<img src="https://static.igem.org/mediawiki/2013/c/c2/Experiment5.png"/>
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<p>Fig 3. No red after night</p>
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<p></p>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.
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<img src="https://static.igem.org/mediawiki/2013/1/10/Experiment6.png"/>
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<p></p>Fig 4.  Constitutive promoter expressing RFP
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<p></p>(Left is 12h, right is 24h)
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<p></p>4)We sent our whole device to companies for sequencing, but it failed.
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Revision as of 05:24, 27 September 2013

HIT-Harbin

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.

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum.

The circuit

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Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum.

The circuit

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum.

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