Part:BBa_K2082152

Promotor, RBS, Stop Beta-Lactamase and Terminator This Part contains a promoter, a ribosomal binding site, a modified beta-lactamse (L79*) and a terminator. This device may be used for characterization and quantification of in vivo mutagenesis systems. Functionality of this part was proven using an error prone DNA-polymerase I. The stop codon in concern was created by a point mutation (T297A). A beta-lactamase containing a stop codon further upstream in the coding sequence can be found here.

Usage and Biology

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 777

After transformation the cells in concern are enabled to grow in chloramphenicol to sustain segregative stability. When mutagenesis has been performed, bacteria is harvested and plated on ampicillin agar. When a mutation reverted the stop codon colonies are formed. The quantity of revertants compared to the total cell number determines the mutation rate. An exact protocol for reversion assays to determine in vivo mutation rates can be found here.

Picture with not growing on ampicillin and growing on chloramphenicol

Figure 1: Possible mutants plated on ampicillin containing agar (right) and chloramphenicol containing agar (left). One of the candidates was used for sequencing and part amplification(red square).

In the first step the beta-lactamase cds of pSB1AK3 was cloned in pSB1C3 containing the other parts of the device by gibson cloning. After verification by plating on ampicillin and chloramphenicol containing agar, one clone was picked and amplified. The plasmid DNA was mutated by using primers containing a mismatch leading to a single point mutation after the first amplification round and a 20 bp overlap similar to the other end of the linear DNA. The DNA has been closed by a one part gibson cloning and transformed to screen for a loss of function (Fig. 1). Single point mutation was verified by sanger sequening.