Difference between revisions of "Part:BBa K3963001"

Revision as of 10:31, 20 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). The trc promotor regulates the expression of the insert behind the lacI with lacIq promoter and a kanamycin selection marker.

Design

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

The cloning was restriction based and without BioBrick prefix and suffix. Therefore the size differs to the registry sequence.

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

Natural transformation in Acinetobacter baylyi ADP1

In Figure 3 it can be seen that after cloning of the pBWB162 plasmid (BBa_K3963000) to the mentioned 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 present.

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 with pit formation can be seen.

beta-agarase YM01-3 activity

We measured the activity of the beta-agarase YM01-3 with the DNS-method which is described in detail on the part page: BBa_K2094002

We measured the activity only in vivo with four replicates.

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 with pit formation 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. The cloned plasmid was still capable with natural transformation mechanism of A. baylyi ADP1 and the beta-agarase activity could also be measured.

Sequence and Features