B2H·RESULT

Our Bacterial Two-Hybrid System (the signal converter) is the core element of our IgEM, so that we decided to construct the IgEM based on the Bacterial Two-Hybrid System.

We begin with the Bacterial Two-Hybrid System from Agilent Technologies.[1]

The Bacterial Two-Hybrid System from Agilent Technologies has the reporter cassata on the F plasmid, which is very inconvenient for alternating the reporter gene. Thus, we constructed a reporter cassata on a plasmid backbone from the Biobrick—pSB4A5. The pSB4A5 has a low-copy repA pSC101-derived replication origin and ampicillin resistance, while pBT is a kind of plasmid that carries a low-copy p15A replication origin and confers chloramphenicol resistance and pTRG carries a low-copy ColE1 replication origin and confers tetracycline resistance. Because they have different origins and different antibiotic resistance, pBT, pTRG and pSB4A5 can co-exist in one bacterial.

pBT: We have designed three kinds of construct on pBT. The first one called pBT-M fuses λcI with multiple cloning sites (MCS). And the second one is a fusion protein named pBT-R containing λcI and RFP. Then we fuse λcI with LGF2 (the GAL4 dimerization domain) to create pBT-L.

pTRG: We have two construct on the pTRG backbone. The pTRG expresses the prey fused to the N-terminal domain of the protein rpoA. One is called pTRG-R, fusing rpoA with RFP, and the other one named pTGR-G is a fusion protein including rpoA and GAL11P (a mutant of GAL11 protein).

pRPT: Reporter cassata is constructed on pSB4A5 with a plac-Or2 promoter and a reporter gene. pRPT-GFP is the construct with GFP as the reporter gene.

LGF2 and GAL11P are proved to have strong interaction with each other and are often used as positive control in different kinds of two-hybrid system. Here, we use pBT-LGF and pTRG-GAL11P to serve as the positive control.

Later on, we acquired the mutant via point mutation, and got the construct of pBT-LGF(H), pBT-LGF(M), pBT-LGF(L). They have different strength of interaction with GAL11P.

1. Three plasmid co-transformation

In order to prove that pBT, pTRG and pRPT can coexist in the bacteria, and at the same time the antibiotics concentration is appropriate, we carried out this co-transformation experiment. The concentrations of the 1000 × antibiotic stock were 35mg/mL for Ampicillin, 50mg/mL for Chloramphenicol and 12.5mg/mL for Tetracycline. We added 1/3 of the normal amount of antibiotic stocks to prepare the three antibiotic LB.

We transformed the plasmid as the following groups:

<p1 style="text-align: center">Table.1 experiment groups of three plasmid co-transformation</p1>

R for pRPT-RFP (Amp), B for pBT-LGF( Cmr), T for pTRG-Gal11P (Tet).

After the transformation, all groups of bacteria were cultured in liquid LB containing three kinds of antibiotics for 72 hours. ⨹RBT and △RBT were respectively cultured in LB with 1 and 1/2 of each basic antibiotic concentration mentioned above. RBT, BT, RT, RB, B, R, T and control were cultured in LB with 1/3 of each basic antibiotic concentration.

<p1 style="text-align: center">Fig.1 The result of three plasmids coexistence experiment</p1>

According to the results, these three plasmids can coexist in the bacteria. We chose LB with 1/4 of each basic antibiotic concentration to screen for the coexistence of three plasmids after transformation and LB with 1/3 of each basic antibiotic concentration to culture the bacteria.

2. Test the Bacterial Two-Hybrid System

We transformed the plasmid as the following groups:

<p1 style="text-align: center">Table2 Experiment groups to test B2H system</p1>

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.

<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 positive control LG (pBT-LGF and pTRG-GAL11P) has significant difference compared to other groups. </p1>

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.

3. Different degrees of interaction and corresponding gene expression

We wanted to know if different degrees of interaction between bait and target 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.

We transformed the plasmid as the following groups:

<p1 style="text-align: center">Table2 </p1>

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.

<p1 style="text-align: center">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</p1>

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 and confirmed that our signal converter could distinguish the different degrees of protein-protein interaction.

References

[1] Ailent Technologies, BacterioMatch II Two-Hybrid System Vector Kit, Instruction Manual,2011.

Team:SYSU-China/file/Project/Result/B2H.html

From 2014.igem.org

Contents

B2H·RESULT

1. Three plasmid co-transformation

2. Test the Bacterial Two-Hybrid System

3. Different degrees of interaction and corresponding gene expression

References