We inserted our gene of interest, HBD, into bacterial cells through cloning and proved the engineering success through colony polymerase chain reaction and sequencing.
HBD Preparation
Using the TM (Melting temperature) calculator, we predicted the annealing temperature of the primer (66°C). However, since we are amplifying HBD directly from EcN (E-Coli Nissle 1917), a biologically complex system, we worried that if the predicted annealing temperature is higher than the actual value, primers would not be able to anneal properly. Hence, gradient PCR, with annealing temperature 66°C , 64°C, 61°C, and 57°C, was utilized to determine the optimal annealing temperature of our primer (Figure 1).
Figure 1. Gradient PCR to determine optimal annealing temperature. The annealing temperature from the leftmost lane to the rightmost lane was set to be 66°C , 64°C, 61°C, and 57°C, respectively. Bands at 888 bp appeared in all four lanes. The letter M is used to indicate the lane loaded with a 1kb DNA ladder.
Gel electrophoresis (Figure 1) confirms the presence of the HBD gene, since bands at 888 bp appeared in all samples. The result proves that 66℃ is the optimal annealing temperature since the band size at 888 bp is the largest for the lane corresponding to 66℃. We thus purified that band and digested it overnight. The concentration of HBD gene after digestion was measured to be 38ng/μL.
Cloning Cycle
We performed mini-prep to isolate the pET28a plasmid from the bacterial cell, washing and purifying the vector with Zymo DNA isolation kit. The plasmids pET28a were then digested overnight. The concentration of the vector after digestion was measured to be 11ng/μL.
Digested pET28a and HBD was then ligated for 2 hours. 0.8 μL of digested HBD and 4.5 μL of digested pET28a were added in the experimental group, while 4.5 μL of digested pET28a were added in the control group. During ligation, the segments, the HBD gene and the pET28a vector, were expected to be ligated by ligase at the restriction enzyme cut sites (EcoR1 & Xho1). The ligated plasmid was transformed into DH5α (competent E. coli cells) , which were then cultured overnight (Figure 2).
Figure 2. Overnight cultures. For both the negative control and experimental group, we plated the bacteria with 50 and 200μL of sample.
The result is inconsistent with our expectations. Normally, we would expect there to be no colonies on the negative control plate since the negative control strain does not carry resistance. On our negative control plate, however, a significant amount of bacteria grew. We still decided to select bacterial strains for colony PCR because we presumed that the colonies on the agar plate all contain pET28a plasmid inserted with the HBD gene.
Colony PCR
We perform colony PCR on 10 randomly chosen colonies from the 200 μL pET28a-HBD plate. We ran gel electrophoresis using these 10 samples (Figure 3).
Figure 3. Result of colony PCR. The letter M is used to indicate the lane loaded with DNA 1 kb ladder.
According to figure 3, the target gene construct (HBD gene in the T7 system), which is 977 bp, did not appear in the region between the 900 bp and 1000 bp mark as expected.
Error Analysis
We believe the error was due to not cutting the plasmid pET28a thoroughly during digestion. This would result in some of the plasmid not ligating with the HBD gene, undergoing self-ligation. To resolve this problem, we thus decided to amplify the plasmid backbone directly via PCR, forcing it to stay in linear form by separating two segments of circular DNA, preventing self ligation. We believed that this could significantly increase the success rate.
New Cloning Cycle
In our new trial, we used the HBD gene from the first trial. The plasmid backbone pET28a, on the other hand, was amplified directly using PCR. Gel electrophoresis, which presented the band at 5369 bp, confirmed the presence of pET28a backbone (Figure 4).
Figure 4. Gel electrophoresis confirms the presence of pET28a. The letter M is used to indicate the lane loaded with DNA 1 kb ladder.
We ligated HBD with pET28a and transformed it into DH5𝛼. The bacterial cells were then cultured overnight (Figure 5).
Figure 5. The agar plates culturing bacteria containing linear vectors-only did not show any colonies, while colonies of the bacteria containing the pET28a plasmid inserted with the HBD gene successfully grew on the plates. The culturing results confirmed our expectation.
Colony PCR
We again performed colony PCR on 10 randomly chosen colonies from the 200 μL pET28a-HBD plate. The gel electrophoresis result, which presented the band at 977 bp, confirmed the presence of the inserted HBD gene in the pET28a plasmid construct.
Figure 6. Result of colony PCR. The letter M is used to indicate the lane loaded with DNA 1 kb ladder.
Sequencing
To ensure that the resulting DNA sequence from PCR matches our designed gene of interest, we sent our construct, pET28a-HBD, to sequencing. The result matches the expected sequence (Figure 7)
Figure 7. The result of sequencing demonstrates a matching rate of 98% out of 1024 bp.
Conclusion
The results of colony PCR and sequencing both demonstrated the success of our cloning cycle. We, therefore, can move on to further steps of our project which consists of confirming the expression of HBD protein through SDS-PAGE.
