Cloning of genes encoding CtPME, CtX157 and CtPMEf into pET28a(+) vector

The pET-28a(+) is a modified form of pBR322 plasmid. It is a frequently used vector for cloning and expression of recombinant proteins in E. coli. pET-28a(+) vector has a strong T7 promoter system originally developed by Studier and colleagues (Studier and Moffatt, 1986; Studier et al., 1990). The expression of genes cloned in pET plasmids is under the control of T7 bacteriophage promoter. The cloned genes are transcribed by T7 RNA polymerase of the host cell. The genes cloned in pET vectors remain transcriptionally silent in the uninduced state. The proteins encoded by the cloned genes are transcribed and contain a His6-Tag, which single step purification using affinity chromatography. The pET-28a(+) vector allows for incorporation of expressed protein with an N-terminal His6-Tag/thrombin/T7-Tag in addition to an optional C-terminal His6-Tag sequence (Fig. 2.3). The location of sequence encoding His-Tag, T7 promoter, T7 terminator, kanamycin resistance and f1 origin are indicated in the Fig. 2.3.

Fig. 2.3 Restriction map of the pET-28a(+) expression vector showing multiple cloning site (158-203 bp), restriction enzyme sites, N-terminal His6-Tag coding sequence (270-287 bp), C-terminal His6-Tag coding sequence (140- 157 bp), T7 promoter (370-386), T7 terminator (26-72 bp), pBR322 origin (3286 bp), kanamycin marker (3995-4807 bp) and a f1 origin (4903-5358).

NheI cuts at 231 and XhoI at 158.

2.2.9.1 Restriction digestion of PCR amplified genes encoding CtPME, CtX157 and CtPMEf and pET-28a(+) plasmid DNA

The pET-28a(+) vector was digested with NheI-XhoI restriction enzymes (Table 2.8). PCR amplified genes encoding CtPME, CtX157 and CtPMEf were also digested with NheI-XhoI to prepare them for ligation with restriction digested pET- 28a(+) vector (Table 2.9). The digestion reactions were incubated in a water bath at 37°C for 90 min. The NheI-XhoI digested pET vector and PCR amplified genes were purified from agarose gel as described in Section 2.2.5.

Table 2.8 Restriction enzyme digestion of pET-28a (+) plasmid DNA.

Table 2.9 Restriction enzyme digestion of PCR amplified genes encoding CtPME, CtX157 and CtPMEf.

Reaction component Gene encoding CtPME (µl)

Gene encoding CtX157 (µl)

Gene encoding CtPMEf (µl)

10x buffer 3.0 4.0 4.0

Nuclease-free water 4.5 13.5 6.5

Bovine serum albumin (10

mg/ml) 0.5 0.5 0.5

PCR amplified product 20 (~125 ng) 20 (~100 ng) 20(~100 ng)

NheI (10 U/µl) 1 (118 ng) 1 (68.7 ng) 1 (15 ng)

XhoI (10 U/µl ) 1 1 13.5

Total 30 40 30

Reaction components Volume (µl)

10x buffer 3.0

Nuclease free water 4.5

Bovine serum albumin (10 mg/ml) 0.5

Plasmid DNA (approx. 13 ng/ µl) 20.0

NheI (10 U/µl) 1.0

XhoI (10 U/µl ) 1.0

Total 30.0

2.2.9.2 Ligation of restriction digested genes encoding CtPME, CtX157 and CtPMEf into pET-28a(+) vector

The NheI-XhoI digested genes encoding CtPME, CtX157 and CtPMEf were ligated into pET-28a(+) vector, which was also digested with same restriction enzymes as described in Section 2.2.9.1. Three ligation reactions were setup using the reaction components mentioned in Table 2.10 and incubated at 16°C overnight to get maximum number of transformants. The reactions were setup at an insert: vector molar ratio of 3:1, where the amount of insert is calculated as mentioned below:

amount of vector (ng) × size of insert (kb) × insert :vector molar ratio = amount of insert (ng) Size of vector (kb)

50 (ng) × 0.42 (kb)

× 3

= 11.7 ng (CtX157)

5.369 (kb) 1

50 (ng) × 1.453 (kb)

× 3

= 40.6 ng (CtPMEf)

5.369 (kb) 1

Table 2.10 Components of reaction for ligating genes encoding CtPME, CtX157 and CtPMEf into pET-28a(+) expression vector.

Reaction component Gene encoding CtPME (µl)

Gene encoding CtX157 (µl)

Gene encoding CtPMEf (µl)

10x Rapid Ligation Buffer 1 1.5 2.5

pET-28a(+) Vector 5 (50 ng) 6.2 (50 ng) 5 (50 ng) Restriction digested product 2.15 (60 ng) 6 (10 ng) 13.5 (15 ng)

T4 DNA Ligase (3 Units/μl) 1 1 1

Nuclease-free water 0.85 0.3 3

Total 10 15 25

50 (ng) × 0.9 (kb)

× 3

= 25.1 ng (CtPME)

5.369 (kb) 1

2.2.9.3 Transformation of ligated recombinant DNA into E. coli TOP10 cells The E. coli TOP10 competent cells were transformed with ligation reactions, after overnight ligation. Preparation of E. coli competent cell preparation has been described in Section 2.2.8. The step-wise transformation protocol is described below:

1. The micro-centrifuge tube containing competent cell (200 µl) was taken out from -80°C and kept on ice for 5 min.

2. 10 µl of ligation mixture was added to cells and the tube was gently tapped 4- 5 times and kept on ice for 30 min. The cells were occasionally tapped gently during 30 min incubation.

3. The cells were given a heat shock at 42°C for 40s.

4. The cells were immediately transferred back to the ice and kept for 5 min.

5. 800 µl of super optimal medium with catabolite repression (SOC) (Hanahan, 1983; Sambrook et al., 1989; given in Section 2.2.7) (previously incubated at 37°C) was added to the transformed cells.

6. The transformed cells were incubated at 37°C in a shaking incubator at 220 rpm for 1h.

7. The cells were centrifuged at 2000g, 25°C for 5 min.

8. 800 µl supernatant was discarded and the cell pellet was re-suspended in remaining 200 µl supernatant.

9. The 200 µl cells were spread plated on LB agar plates as described in Section 2.2.6.1 supplemented with antibiotics. The LB agar plates were incubated overnight at 37°C.

10. The transformation efficiency was calculated using the following formula, Transformation

efficiency = No. of colonies on LB plate

= cfu/µg Amount of insert (µg)

The 15-20 µl of ligation mixture was added to 200 µl E. coli TOP10 competent cells, following the above transformation protocol. The transformed TOP10 cells were plated on LB plates supplemented with kanamycin (50 µg/ml) and grown overnight at 37°C, 180 rpm.

2.2.9.4 Isolation of plasmid DNA from transformed colonies by miniprep kit Overnight incubated plates were observed for colonies. Colonies preferably from the centre of the plate were randomly picked in a laminar air flow and grown overnight in 5 ml LB medium supplemented with kanamycin (50 µg/ml). The plasmid DNA from this 5 ml culture was isolated by miniprep kit (Sigma-Aldrich, USA) following the protocol mentioned in Section 2.2.9.4.1.

2.2.9.4.1 Plasmid isolation protocol by miniprep kit

1. 10 ml from each of the grown culture containing recombinant plasmid were pelleted in 1.5 ml microcentrifuge tube aseptically.

2. The cells were then centrifuged at 14000g for 1 min and the process was repeated six times with 1.5 ml culture (Total 9ml culture).

3. The resulting cell pellet of each recombinant derivative was re-suspended in 200 μl resuspension solution and vortexed. RNAse at final concentration of 0.3 mg/ml was added to the re-suspension solution prior to use.

4. 200 μl of lysis solution was added to each tube and the tubes were inverted gently 5-6 times to ensure mixing and allowed to stand for 2-5 min.

5. 350 μl of neutralization solution was added to the mixture and the tubes were inverted again for 4–6 times to mix properly.

6. The mixture was centrifuged at 16,000g for 10 min.

7. The DNA binding columns were prepared and activated by adding 500 µl of column preparation solution to binding column and centrifuging at 14,000g for 1 min. The flow through accumulated in collection tube was discarded.

8. The clear lysate was then transferred to activated DNA binding column, centrifuged at 14,000g for 1 min and the flow through in the collection tube was discarded again.

9. The plasmid DNA bound to the column was washed with wash solution and spun at 14,000g for 1 min. The flow through was discarded and the column was given another 1 min spin at 14,000g for removing the wash solution completely.

10. The DNA binding column was transferred to a fresh sterile microcentrifuge tube and 30 µl of TE buffer solution or DNAse free water was added at the centre of binding column. The microcentrifuge tube was allowed to stand for 10 min at room temperature and then plasmid DNA was eluted by centrifugation at 14,000g for 1 min.

11. The eluted plasmid DNA in sterile microcentrifuge tube was stored at -20°C.

2.2.9.5 Screening of recombinant plasmid DNAs for positive clones by restriction digestion

15 µl of recombinant plasmids from pET-28a(+) clones of CtPME, CtX157 and CtPMEf that were isolated in the last step, were taken in separate fresh sterile micro-centrifuge tubes for restriction enzyme digestion analysis. The recombinant plasmid DNA of each of the above mentioned derivatives was digested with restriction enzymes, NheI and XhoI, to check for positive clones following a 30 µl reaction mixture set up as mentioned in Table 2.8.

In document PhD Thesis by Vikky Rajulapati (Page 83-90)