Temperature sensitive mutant of the broad host range plasmid pBBR1MCS-2

Broad host range plasmid pBBR1MCS-2 derived from pBBR1 is mobilizable and compatible with various plasmid incompatibility groups and wildly used for cloning and expression in laboratory. However, temperature sensitive (Ts) mutations of pBBR1MCS-2 are rarely reported. In this study, the rep gene (involved in replication) and its promoter and terminator were mutated by error prone PCR. The resulting PCR product was inserted into the backbone of pBBR1MCS-2 using the Gibson assembly kit. One mutant plasmid (pBBR1MCS-2-Ts) was identified that could be maintained in Escherichia coli T1, DH5α, BW25113, S17-1 and Rhodobacter sphaeroides when grown at low temperature but was lost from host cells grown at high temperature in the absence of antibiotic selection. Sequence analysis indicated that pBBR1MCS-2-Ts had three points mutation: a mutation of T to A at nucleotide number -43(relative to translation start site of rep); a mutation of A to G at nucleotide number 73 resulting a codon change of Lys to Glu at residue number 25; a mutation of A to G at nucleotide number 436 resulting a codon change of Ile to Val at residue number 145. Further point mutation revealed that the mutation of Lys to Glu at residue number 25 causes the plasmid replication to be temperature sensitive. The Ts and the wild type of replication proteins (REPs) were expressed and purified. Circular Dichroism spectra suggest that the secondary structure of Ts-REP was changed and the T_m (temperature at which 50% of denaturation is achieved) has reduced by 4.9 ℃.