Part:BBa_K4908003

AutolysinCBD-pET28a

AutolysinCBD-pET28a

Usage and Biology

The target gene fragment (AutolysinCBD) comes from the C-terminal cell wall binding domain of Bacillus cereus ATCC 14579 autolysin. Bacillus cereus, also known as cactus bacillus, is a Gram-positive bacterium, β Hemolytic rod-shaped bacteria. Often found in soil and food, some strains can cause Fried Rice Syndrome[1]. This fragment is a characteristic sequence of Bacillus cereus.

Construction Design

The part consists of two parts: the autolysinCBD gene fragment (BBa_K4908001 Part: BBa K4908001 - parts.igem.org) and the pET28a vector backbone (BBa_K3521004 Part: BBa K3521004 - parts.igem.org).

In order to study the colorimetric of Bacillus cereus (BC) binding proteins labeled with anti-His-HRP antibody and TMB substrate, we aimed to construct a His-tagged BC autolysin cell wall binding domain (CBD) with the vector pET-28a.

The target gene fragment CBD sequence comes from the C-terminal cell wall binding domain of Bacillus cereus ATCC 14579 autolysin. Bacillus cereus, also known as cactus bacillus, is a Gram-positive bacterium, β Hemolytic rod-shaped bacteria. Often found in soil and food, some strains can cause Fried Rice Syndrome.2 The fragment we used is a characteristic sequence of BC and was synthesized by the company Genscript.

The experimental design of this study is as follows:

1. Construction of plasmids pET28a-His-autolysinCBD
2. Expression and purification of autolysinCBD
3. Detection system optimization
4. Colorimetric detection with autolysinCBD

Fig. Map of pET-28a-autolysinCBD

Experimental Approach

We plan to use homologous recombination to obtain the recombinant plasmid pET28a-autolysinCBD. First, we digest the pET28a vector backbone using NdeI and xhoI while obtaining a large number of target fragments autolysinCBD 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.

Figure 1 Construction of plasmid pET28a-autolysinCBD

Test 1: PCR of bacterial fluids

We picked recombinants grown on solid plates for bacteriophage PCR and the results are shown below. Two recombinant plasmids of pET28a-autolysinCBD appeared with correct bands, and the preliminary validation results indicated that our constructs were successful.

Figure 2 PCR of bacterial fluids

Test 2: 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.

Figure 3 SDS-PAGE

Test 3: 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%).

Figure 4 Optimal reaction conditions for detection systems

Test 4: 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.

Figure 5 Results of testing different concentrations of bacterial fluids

Learn

To detect BC pathogens using a colorimetric assay based on smartphone and bacterial binding proteins, we first recombinantly expressed and purified a bacterial binding protein (His-autolysinCBD). Per the results mentioned above, we can validate our theory as a success of the colorimetric detection by the binding proteins of BC. In order to implement our detection method, we need to conduct more experiments, including investigating the limit concentration of detection and the quantity relationship between RGB values of the test samples and the bacteria concentrations per the app development requirement.

Reference

[1] Ivanova Natalia, Sorokin Alexei, Anderson Iain, Galleron Nathalie, Candelon Benjamin, Kapatral Vinayak, Bhattacharyya Anamitra, Reznik Gary, Mikhailova Natalia, Lapidus Alla, Chu Lien, Mazur Michael, Goltsman Eugene, Larsen Niels, D'Souza Mark, Walunas Theresa, Grechkin Yuri, Pusch Gordon, Haselkorn Robert, Fonstein Michael, Ehrlich S Dusko, Overbeek Ross, Kyrpides Nikos. Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis.[J]. Nature, 2003, 423(6935)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4804
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 3024
  Illegal NgoMIV site found at 3184
  Illegal NgoMIV site found at 4772
  
• 1000
  COMPATIBLE WITH RFC[1000]