Difference between revisions of "Part:BBa K3963001"

Latest revision as of 15:53, 21 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 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).

Figure 2 Plasmid pBAV1k-lacI-Trc-beta-agarase YM01-3 characterization. In (A) the size of pBAV1k-lacI-Trc-beta-agarase YM01-3 is shown in a gel. (B) shows the preperated plasmids cloned into E. coli DH5ɑ. (C) represents the sequencing data from all four plasmids. All plasmids are aligned to the designed plasmid.

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 in vivo with four replicates for the positive control (A. baylyi NT beta-agarase) and two A. baylyi wildtype (WT) negative controls.

Figure 4 β-agarase “in vivo” DNS-method in A. baylyi. (A) shows the tubes with the DNS reaction solutions. The graph in (B) represents the measured solutions at 540 nm regarding the standard curve. We compared negative control A. baylyi without a β-agarase (WT) in comparison to cloned A. baylyi with the pBAV1k-lacI-Trc-beta-agarase YM01-3 plasmid (NT agarase).

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.

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