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
The cloning was restriction based and without BioBrick prefix and suffix with the plasmid backbone part BBa_K3963003. 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).
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.
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 in vivo with four replicates for the positive control (A. baylyi NT beta-agarase) and two A. baylyi wildtype (WT) negative controls.
To measure the exact amount of the reducing sugars in the concentration of mg/ml, in the following table the x-values from the Figure 4B are demonstrated.
| Sample | Absorbance Value | total amount of reducing sugars(mg/mL) |
|---|---|---|
| A. baylyi negative control | 0.184∓0.003 | 0.038 |
| A. baylyi in vivo positive control | 0.233∓0.015 | 0.077 |
Discussion
The line for the plasmid pBAV1k-lacI-Trc-beta-agarase YM01-3 in our agarose gel appears a bit too low (instead of at 5.5 kb the line is seen at 4 kb). This could be due to the circular structure of the plasmid, but the ladder consists of linear DNA. Especially for longer DNA fragments, circular DNA travels faster through the gel than linear ones. The reason for that is because circular DNA can coil around itself and does not get stuck in small agar pores as linear DNA does. However, the sequencing data in Figure 2C indicate that in all preparated plasmids the beta-agarase YM01-3 is successfully transformed. A visual indication for the beta-agarase YM01-3 presence is 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 (see Fig. 3) and the beta-agarase activity could also be measured (see Fig. 4). The efficiency of the beta-agarase YM01-3 was low but still detectable.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 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]
| None |