Course Code: MBTE 4033
Course Name: Methods in Molecular Biotechnology Lab I
Student Name: Cheng Kai Wen
Construction of Recombinant DNA Encoding Luciferase Protein Reporter and Transformation
Luciferase, abbreviated as “Luc”, refers to the enzymes capable of producing bioluminescence. The segment of DNA encoding luciferase was used as the insert in this study. The objective of the study is the construction of recombinant DNA, encoding luciferase protein reporter, and the transformation of the recombinant DNA into the E. coli. In this study, we selected pET507a, a modified pET3-d vector, as the vector to complete subcloning process.
The procedure was as described in the manual of Methods in Molecular Biotechnology Lab I.
Results and Discussion
Before the conduction of PCR on pTA-Luc, the primers for PCR need to be designed beforehand. The corresponding primers will anneal to the both ends of target segment. In the experiment, the iProof DNA polymerase will attach to the primers, and multiply the target sequence after the PCR reaction. Figure 1 shows the sequences of the primers used in the PCR reaction.
Figure 1. Sequences of forward and reverse primers
The purpose of PCR is to multiply the target sequence on pTA-Luc into huge number of copies, leading to the amplification of DNA insert. The two PCR tubes were labelled as S1 and S2 respectively, identically containing primers, iProof DNA polymerase, dNTPs, pTA-Luc, 5X iProof HF buffer and water. The PCR result was checked through electrophoresis and observed with transilluminator. The photograph was taken as Figure 2.
|Figure 2. Photo of electrophoresis loaded with PCR result. KB as kilo base pairs; MW as DNA ladder; S1 and S2 as PCR results from S1 and S2 PCR tubes respectively; +ve as positive control provided by the laboratory demonstrator.|
The photograph showed that the PCR result was successive. Lines from S1, S2 and positive control were all containing clear bands corresponding to the size of approximately 1.7 kilo base pairs on the DNA ladder, showing that DNA from S1, S2 and positive control lines were having the similar amount of DNA.
Gene Clean and Ligation
The insert from PCR result previously was then purified with centrifugation, using QIA quick PCR purification kit, and then mixed with, 10X NEBuffer 4, restriction enzymes SacI and KpnI and water, and labelled as tube 5. Another mixture of pET507a vector and the same restriction enzymes was made as well, labelled as tube 1. To ensure that the samples in both tubes are digested correctly by the restriction enzymes, an electrophoresis was performed.
Gene clean refers to the action of rescuing DNA from the gel after electrophoresis. The result of gene cleaning was shown in Figure 3.
|Figure 3. Photo of electrophoresis loaded with gene cleaning result. Sample 1 as the insert digested with restriction enzymes originally from tube 1; sample 5 as the pET507a vector digested with restriction enzymes originally from tube 5; ladder as DNA ladder; KB as kilo base pairs.|
Figure 3 shows that we successfully rescued the DNA from the gel, although the size read from the ladder are different with figure 1. The difference was resulted from the loosing of part of the insert during restriction enzyme digestion.
After checking the gene clean result, three tubes were labelled L1, L2 and L3. L1 tube contained insert and vector; L2 tube contained insert and vector both provided by laboratory demonstrator; and L3 tube contained only vector. The ligation was done automatically when the identical sticking ends, resulted from restriction enzymes digestion, on insert and vector bind to each other in the tube L1 and L2 during this process.
Five tubes containing competent E. coli cells and five LB agar plates, containing ampicillin, were labelled as T1, T2, T3, T+ve and T-ve respectively. T1, T2 and T3 tubes were added with the corresponding ligation reaction from L1, L2 and L3 tubes, whilst T+ve was added with pET507a vector only, and T-ve was added with water. The mixture of each T tube was then undergoing through heat shock and cold shock process, for allowing the entry of ligation product into the competent cell. After that some LB solution was added to each tube to allow recovery, and the contents from each T tube was then pipetted to the corresponding T LB agar plates.
After growing overnight, the number of colonies was counted as figure 4.
Figure 4. Number of colonies from each T LB agar plates with transformed E. coli
pET507a contains capability of ampicillin resistance. Therefore, they can be grown on the LB agar plates, which contain ampicillin.
Confirmation of the recombinant DNA
Some E. coli cells were picked from the colony from the T LB agar plates, and transferred to the corresponding M tubes. After the minipreparation process, some E. coli cells from M tubes were transferred to the corresponding R tubes, together added with restriction enzymes. Some PCR tubes were added with primers, water and PCR master mix, then labelled with P1, P2, P3 each additionally added with diluted contents from corresponding M tubes. Furthermore, pET507a-Luc containing PCR tube was also prepared. Altogether, electrophoresis was performed on tubes hereby mentioned, and the result was obtained as figure 5.
|Figure 5. Photo of electrophoresis for recombinant DNA confirmation. R1, R2 and R3 contain digested recombinant DNA; M1, M2 and M3 contain undigested DNA; R+ve contains digested pET507a plasmid; M+ev contains undigested pET507a plasmid; P1, P2 and P3 contain undigested DNA, PCR master mix and primers; -ve as autoclaved water; KB as kilo base pairs.|
From figure 5, R+ev line revealed the band of the vector pET507a, and P1, P2, P3 and pET507a-Luc lines revealed the band of the insert luciferase. As we could find corresponding bands of both vector pET507a and luciferase insert on R1, R2 and R3 lines. We can conclude that the transformation experiment was a success.