Difference between revisions of "Part:BBa I12210:Experience"

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Applications of BBa_I12210

In 2014, SYSU-China applied BBa_I12210 into Integrated Evolution Machine (IgEM). It was construted into the Bacterial Two-Hybrid System. BBa_I12210(plac Or2-62) was incorporated into the plasmid pRPT, which is for the reporter cassata. The plasmid pRPT uses pSBA45 backbone with a low-copy repA pSC101-derived replication origin and ampicillin resistance.The reporter here is GFP. It is shown as the following:

Fig.1 pSB4A5-Or2-RBS-GFP

The biology function of BBa_I12210 was tested with the Bacterial Two-Hybrid System. And here are some experiments we conducted to test the Bacterial Two-Hybrid System. The results show that BBa_I12210 can undertake the function of binding site for lambda cI. And it is not inducible by IPTG.

Test the Bacterial Two-Hybrid System

We transformed the plasmid as the following groups:

Table 1 Experiment groups to test B2H

Then the bacteria culture of these groups were measured by Bio-Tek Synergy Hybrid Reader. The excitation wavelength was 510nm, while the emission wavelength was 550nm.

Fig.2 Fluorescent intensity measured by Bio-Tek Synergy Hybrid Reader with 510nm excitation wavelength and 550nm emission wavelength. The positive control LG (pBT-LGF and pTRG-GAL11P) has significant difference compared to other groups.

The figure showed that there was a significant difference between the positive control of LG and the other experimental groups. The fluorescence intensity of the positive control was nearly 5 times larger than the ones of the other groups. RFP didn’t have any influence on the expression of GFP. The independent expression of LGF2 and GALP11P also affected the expression of GFP without conspicuousness.

Different degrees of interaction and corresponding gene expression

We wanted to know if different degrees of interaction between LGF2 and GAL11P could be detected by the expression of reporter gene, so we conducted a test for it. According to Patricia et al.2001, we used the technique of Site-Directed Mutagenesis (SDM) to get three mutants of LGF2. They are pBT-LGF(L)E75A (the corresponding plasmid is marked as L), D78A (pBT-LGF(M)) and R74A (pBT-LGF(H)). And they respectively have low, medium and high level of interactions with GAL11P. The reporter gene here is GFP.

We transformed the plasmid as the following groups:

Table2

Then the bacteria culture of these groups were measured by Bio-Tek Synergy Hybrid Reader. The excitation wavelength was 510nm, while the emission wavelength was 550nm.

Fig.3 Fluorescent intensity measured by Bio-Tek Synergy Hybrid Reader with 510nm excitation wavelength and 550nm emission wavelength. Different mutants of the LGF have different strength of interaction with GAL11P. The LGF (L) has low binding affinity

The result indicated that there were considerable differences among the expression of GFP activated by L, M and H. The fluorescence intensity of H group was much larger than that of M group and M was also larger than L. This was consistent with the result in the reference.

For more information please visit our wiki

http://2014.igem.org/Team:SYSU-China/content.html#Project/Result/B2H

References:

[1]Simon L. Dove et al. Activation of prokaryotic transcription through arbitrary protein-protein contacts. Nature. 1997,386: 627-630.

[2] Patricia Hidalgo et al, Recruitment of the transcriptional machinery through GAL11P: structure and interactions of the GAL4 dimerization domain, GENES & DEVELOPMENT, 2001, 15:1007–1020.

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