Part:BBa_K1333200

lambda cI-LGF2(E75A)

Fuse lambda cI with LGF2 mutated from Glu to Ala at site 75 (the Glu which locates after three continuous Ala behind lambda repressor is at site 58). As one of the components in Bacterial Two-hybrid System, it causes little interaction with GAL11P compared to the normalized WT.

Usage and Biology

lambda cI

λcI is the full-length bacteriophage lambda repressor protein containing 237 amino acids. It is composed of the animo-terminal DNA-binding domain and the carboxyl-terminal dimerization domain. It can bind to a specific DNA sequence called λ operator or Or2 sequence.

LGF2

LGF2 is the GAL4 dimerization domain, which is proved to have strong interaction with GAL11P. Both LGF2 and GAL11P are often used as positive control in different kinds of two-hybrid system. The mutant of LGF2(E75A) should have low level of interaction with GAL11P compared to normalized WT.

lambda cI-LGF2

Under the condition of normalized WT, LGF2 is fused to the C-terminal domain of λcI and GAL11P is fused to the N-terminal domain of rpoA. Since LGF2 and GAL11P interact strongly enough, rpoA is recruited at the promoter and activate transcription of reporter gene.

Fig.1 lambda cI-LGF2 and its interaction with GAL11P

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 712
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 767
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

How the mutant of LGF2(E75A) functions

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:

Table1

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. 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.

family	with BBa_K1333201, BBa_K1333202, BBa_K1333203
function	hardly interact with Gal11P
protein	fusion protein of lambda cI and LGF2