Part:BBa_K3285334

mutD5 under pBAD promoter

The gene mutD codes for epsilon subunit of DNA polymerase III. Epsilon subunit is responsible for 3’-5’ proofreading activity of DNA polymerase III. The gene mutD5 is a dominant-negative mutant of mutD, which codes for faulty epsilon subunit. So when expressed, the faulty epsilon subunit gets incorporated into DNA polymerase III which compromises the proofreading activity of DNA polymerase III. Which in turn increases the mutation rate of bacteria.

This composite promoter consists of 2 parts that pBAD-araC promoter and include (Fig.1)

Fig.1: The basic gene circuit of the mutator system

USAGE AND BIOLOGY

In the presence of arabinose, a mutD5 will get expressed increasing the mutation rate of bacteria.

CONSTRUCTION

Restriction free cloning (PCRs) was used for obtaining the final composite part and
intermediate parts are described below.

Fig.2: Amplification of pbad

Fig.4: Amplification of mutD5

Fig.3: Stitching of mutD5 and pBAD

araC-pBAD amplified from biobrick(BBa_I0500). For amplification of araC-pBAD from a plasmid, 45 ng of template was used. annealing temperature (A.T) was 56 deg used. The extension period (E.P) was 75 sec(Fig.2) The resulting bright bands obtained in each case were gel extracted and purified(fig2).

mutD5 is synthesized from twist bioscience. For amplification 50 ng of template was used. Annealing temperature was 58 deg used. The extension period was 50 sec. The band of interest was gel extracted and purified(fig3).

The two genes were used as a template and a 3-step overlap extension PCR along with primers in a pot reaction was performed to stitch them together at an A.T of 58 deg. and an E.P of 145 sec. About 50 ng of pBAD was used and equimolar quantity of mutd% was used. The resultant product was gel extracted and purified(fig4).

Then stitched product(pBAD + mutD5) was inserted into pSB1C3 vector using 2A assembly and transformed into DH5-Alpha cells. Then clone was confirmed using colony PCR.