Supplementary Material S1: Strategy to construct barnase gene with PstI site
A PstI site was introduced in the barnase gene by mutating the ‘T’ base at the 45th
position to a ‘G’ creates the site ‘CTGCAG’. Further, CTT and CTG both encode for the
same amino acid, i.e. lysine. In the present study we modified barnase gene sequence by
creating two adjacent PstI sites separated by four nucleotides so that the modified
barnase gene is not able to synthesize any functional protein. This was done for ease in
cloning of the modified barnase gene without the need to express the barstar protein. The
modified barnase gene was synthesized by Splicing by overlap extension (SOE) method
(Horton, 1993). This PCR method is used either to insert mutation at specific positions or
to splice smaller DNA fragments into a single larger fragment. The overall strategy is
outlined in the figure below. The gene was synthesized in two parts. The initial 63 base
pairs were made by annealing the sense and the antisense strand which were
commercially synthesized. The sequence of the oligonucleotide strands and primers is
given in the Table below. This fragment also carried the base change and the additional
10 base pairs to create the two PstI sites. The rest of the gene was amplified using a
forward and a reverse primer F1 and R1. The F1 primer carried a region overlapping with
the fragment synthesized by annealing. The two fragments were assembled by SOEing PCR and the product was finally amplified by using primers F2 and R2. The amplified
product of 346bp was cloned in pCR® Blunt-II-TOPO® vector and then sequenced.
Table: Sequence of oligonucleotides and primers used for the synthesis of modified
barnase by (SOE) PCR.
S.No.
Oligonucleotide/Primer
Sequence (5'-3')
1.
Sense strand
5'-AAT CCA TGG CAC AGG TTA TCA ACA
CGT TTG ACG GGG TTG CGG ATT ATC TGC
AGA ATT CTG CAG-3'
2.
Antisense strand
5’-CTG CAG AAT TCT GCA GAT AAT CCG
CAA CCC CGT CAA ACG TGT TGA TAA CCT
GTG CCA TGG ATT-3‘
3.
Forward primer (F1)
5'- GAT TAT CTG CAG AAT TCT GCA GAC
ATA TCA TAA GCT ACC-3'
4.
Reverse primer (R1)
5'- ATA TCT AGA TTA TCT GAT TTT TGT
AAA GGT CTG ATA ATG GTC CG-3'
5.
Forward primer (F2)
5'-GCT CCA TGG CAC AGG TTA TCA ACA
CGT T-3'
6.
Reverse primer (R2)
5'- ATA TCT AGA TTA TCT GAT TTT TGT
AAA GGT CTG ATA ATG GTC CG-3'