Part:BBa_K3963001
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
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 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 existing.
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
- 10INCOMPATIBLE WITH RFC[10]Unknown
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2292
Illegal BamHI site found at 3167 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 3183
Illegal AgeI site found at 3220 - 1000COMPATIBLE WITH RFC[1000]
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