Revision as of 11:51, 11 October 2023

MS18-pET28a

MS18-pET28a

Contribution by Team SubCat-Shanghai 2023

Usage and Biology

The gene fragment MS18 we used comes from the tail fiber protein (TFP) MS18 of Vibrio parahaemolyticus phage VPMS1. Vibrio parahaemolyticus is a marine bacterium that mainly comes from seafood such as fish, shrimp, crab, shellfish, and seaweed. Consumption of food containing this bacterium can lead to food poisoning, also known as halophilic food poisoning. Clinically, acute abdominal pain, vomiting, diarrhea, and watery stools are the main symptoms[1]. This gene is a characteristic fragment of Vibrio parahaemolyticus.

Construction Design

We ligated a segment of the MS18 gene sequence (BBa_K4908000 Part: BBa K4908000 - parts.igem.org) to the original part (BBa_K3521004 Part: BBa K3521004 - parts.igem.org) to obtain the new part (BBa_K4908002).

In this project, we developed a detection system to detect bacterial concentration using the Vibrio parahaemolyticus phage VPMS1 tail fiber protein (TFP) MS18.

Experimental Approach

We plan to use homologous recombination to obtain the recombinant plasmid pET28a-MS18. First, we digest the pET28a vector backbone using BamHI and xhoI while obtaining a large number of target fragments MS18 attached to the homology arm by PCR. Then, we purified and recovered the vector and target fragments by agarose gel electrophoresis to improve the success rate of recombination. Based on previous experience, we decided to use Vazyme's C115 kit for homologous recombination. Subsequently, we transformed the recombinant product into E. coli sensory state and cultured it overnight at 37°C. On the next day, it was observed that colonies grew on the plate, and we picked some colonies for bacteriophage PCR. After initial verification of the experimental results, we sent the successfully constructed plasmid to the company for sequencing to ensure that we had obtained the correct recombinant plasmid.

Test 1: SDS PAGE

In order to obtain the target protein, we need to add an additional chemical reagent, IPTG, so as to induce protein expression. After inducing expression overnight, we extracted the proteins from the cells by high-speed centrifugation and ultrasonic crushing. Subsequently, in order to obtain high purity of the target proteins, we performed protein purification using a nickel column. After purification was completed, SDS PAGE analysis was performed in order to verify the success of our experiments.

Test 2: Detection System Optimization

We plan to detect bacterial concentration by enzyme-linked immunosorbent assay. The principle of this method is the specific binding of antibody and antigen, and the results need to be obtained by observing the color change of the TMB chromogenic solution. Therefore, we need to study the two experimental conditions in the detection system to obtain the optimal reaction conditions. These two reaction conditions are the enzyme-catalyzed reaction time and different concentrations of TMB color development solution. We set up two different sets of experimental conditions, one varying the enzyme-catalyzed reaction time (0h/0.5h/1h/1.5h/2h) and one varying the reaction concentration of TMB (0.01%/0.02%/0.03%/0.04%/0.05%).

Test 3: Detection of Different Concentrations of Bacterial Fluids

We serially diluted overnight bacterial cultures (Bacillus cereus, Vibrio parahaemolyticus) to 102-107 CFU/mL using PBS, and subsequently added 1 mL of each concentration to a sterile centrifuge tube for determination. The reaction conditions used in this experiment were the optimal reaction conditions obtained from previous experiments.

Reference

[1] Ramírez-Orozco Martín, Serrano-Pinto Vania, Ochoa-Álvarez Norma, Makarov Roman, Martínez-Díaz Sergio F. Genome sequence analysis of the Vibrio parahaemolyticus lytic bacteriophage VPMS1.[J]. Archives of virology, 2013, 158(11)

Sequence and Features