Part:BBa_K1333204

Or2-RBS-GFP-terminator

This is a reporter which combines plac Or2-62 and the generator of GFP. In the beginning of the Or2 sequence, there is a binding site for protein lambda cI called lambda cI operator. In Bacterial Two-hybrid System, the protein lambda cI is linked to a molecular called bait. And the corresponding target is linked to rpoA, which is the α subunit of RNA polymerase. The Or2 is reponsible for the recruitment of lambda cI-bait. When the bait interacts with the target, the rpoA is carried to downstream promoter and activates the downstream expression which is GFP in this case. Stronger interactions between baits and targets indicate higher concentration of rpoA near the promoter and higher level of transcription of GFP. Hence, we can tell the interactional degree between baits and targets from the fluorescence intensity of GFP.

Fig.1 lambda cI operator and the downstream GFP reporter

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 741
  

Functional Parameters

BBa_K1333204 is the most important part in the Bacterial Two-Hybrid System. And its effectiveness was tested in the whole Bacterial Two-Hybrid System as the following experiments. It is on the backbone of pSB4A5. The plasmid is shown here.

Fig.2 pSB4A5-Or2-RBS-GFP

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.

color

fluorescent green