Difference between revisions of "Part:BBa K3963001"

Revision as of 13:46, 19 October 2021

pBAV1k-lacI-Trc-beta-agarase YM01-3

The plasmid is combined by the backbone from the pBWB162 plasmid (BBa_K3963003) and cloned by restriction based cloning with the insert beta-agarase YM01-3 (BBa_K2094002). mCherry protein was cut out. The plasmid still contains the lacI behind lacIq promoter and a kanamycin selection marker.

Design

Figure 1 Plasmid pBAV1k-lacI-Trc-beta-agarase YM01-3 design. The plasmid size is 5560 bp.

Experiments and results

Plasmid characterization

The plasmid should have a size of 5560 bp but in the gel the plasmid looks smaller about 4 kb (see Fig. 2A).We transformed the plasmid pBAV1k-lacI-Trc-beta-agarase YM01-3 into E. coli DH5ɑ and the typical pit formation can be observed (see Fig. 2B). We send the plasmid to sequencing to control the beta-agarase insert (see Fig. 2C).

Figure 2

Natural transformation function

In Figure 3 it can be seen that after cloning of the pBWB162 plasmid (BBa_K3963000) to the recent plasmid pBAV1k-lacI-Trc-beta-agarase YM01-3 the ability to be taken up by Acinetobacter baylyi ADP1, a natural competent bacterial strain, is still existing.

Figure 3 Plasmid pBAV1k-lacI-Trc-beta-agarase YM01-3 transformed in A. baylyi ADP1. In the picture about 8 transformed colonies of A. baylyi can be seen.

Discussion

The plasmid pBAV1k-lacI-Trc-beta-agarase YM01-3 appears in the gel a bit too small (instead of 5.5 kb at 4 kb). This could be due to the circular structure of the plasmid whereas the ladder is linear DNA. However, the sequencing Data in Figure 2C indicate that in all preparated plasmids the beta-agarase YM01-3 is successfully transformed. Another hint are the pit formations of the E. coli DH5ɑ in Figure 2B. At last, the cloned plasmid was still capable with natural transformation mechanism of A. baylyi ADP1.

Sequence and Features