Difference between revisions of "Part:BBa K3285334"
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Restriction free cloning (PCRs) was used for obtaining the final composite part and <br> intermediate parts are described below. | Restriction free cloning (PCRs) was used for obtaining the final composite part and <br> intermediate parts are described below. | ||
| − | araC-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. |
Revision as of 20:27, 21 October 2019
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.
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.