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From 2014.igem.org

Integration·RESULT

Bacterial Two-Hybrid System integrate with RNAT

We construct plac-Or2-FourU-GFP on the pSB4A5 for the test.So that we can see whether FourU can control GFP translation in a temperature dependent manner.

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<p1 style="text-align: center">Map 1 construct of plac-Or2-FourU-GFP</p1>

We co-transformed the plasmid as the following groups:

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These four groups of bacteria were cultured at 30℃ and 37℃ respectively. All the OD was modified to be the same before the sample was tested by 510nm as excitation wavelength and 550nm as emission wavelength.

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<p1 style="text-align: center">Fig.1 Fluorescent intensity measured by Bio-Tek Synergy Hybrid Reader with 510nm excitation wavelength and 550nm emission wavelength. The group in 37℃ is significantly higher that the groups in 30℃.</p1> <a class="fancybox" rel="group" href=" "><img src=" " style="width:800px"alt="" /></a> <p1 style="text-align: center">Fig.2 Fluorescent intensity measured by Bio-Tek Synergy Hybrid Reader with 510nm excitation wavelength and 550nm emission wavelength. The difference among 30℃ are not significant while the difference among 37℃ is significant.</p1>

2.Bacterial Two-Hybrid System integrated with M13

The reporter gene on pRPT is gene-Ⅷ from M13 bacteriophage. The corresponding plasmid is named pRPT-gⅧ.

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After the co-transformations as follows, we tested if gene Ⅷ can be expressed in Bacterial Two-Hybrid System.

LB with 1/4 of each basic antibiotic concentration was used to screen for the bacteria containing all three plasmids in each group. Then liquid LB with 1/3 of each basic antibiotic concentration was used to culture the bacteria for 24 hours before the OD was modified to be consistent. After that, the bacteria were broken down and centrifuged. The intact bacteria, the supernatant and sediment from the centrifugation were all prepared for samples of Western Blot. gene Ⅷ was fused to FLAG for the following Western Blotting analysis.