Plasmid

Part:BBa_K3963000

Designed by: Silja Malkewitz   Group: iGEM21_Heidelberg   (2021-09-30)


pBWB162

pBWB162, designed by Keith Tyo [1] is based on the pBAV1k origin of replication which is based on the pWV01 cryptic plasmid was originally purified from Streptococcus cremoris [2]. It was used as a suitable plasmid for natural transformation in Acinetobacter baylyi ADP1 (A. baylyi). This plasmid contains an mCherry protein behind a trc promoter, lacI behind lacIq promoter and a kanamycin selection marker.

Experiments and Results

Plasmid pBWB162 expression

We grew the ordered Plasmid in Escherichia coli (E. coli) DH5α on agar plates and prepared plasmid preparation with the QIAGEN® Plasmid Plus Midi Kit (Fig.1A). We transformed this plasmid by natural transformation protocol successfully in A. baylyi ADP1 according to the protocol from Biggs et. al 2020 [3]. We observed an IPTG inducing mCherry expression (Fig.1B).

Figure 1. Plasmid pBWB162 size and mCherry expression in A. baylyi ADP1. (A) shows the prepped plasmid from E. coli DH5α with the size of 5013 bp and (B) represents the mCherry expression in A. baylyi ADP1 pellet in an overnight culture induced through IPTG.

Natural transformation efficiency in monoculture

We further calculated the natural transformation efficiency. We transformed the plasmid pBWB162 and lacI-PT5-gusA-pBAV1k designed by Ichiro Matsumura [4] into A. baylyi ADP1 and counted the colony forming units (CFU). We compared two different protocols (see Fig. 2E). The first protocol is called “log-phase” because the A. baylyi ADP1 were first incubated for 5 hours to reach the exponential phase and then the plasmid was added for 3 hours. In the second protocol called “overnight” the bacteria were diluted from the overnight culture and the plasmid was added directly for 5 hours and then the cultures were streaked out. We choose 25 ng and 100 ng of plasmid because the literature was not clear.

Figure 2. Efficiency of natural transformation in A. baylyi ADP1. (A-D) shows the natural transformation plates.(A) is the positive control on a non-selective plate, (B) is the negative control without any plasmid DNA, (C) is the natural transformation with 25 ng of the pBWB162 plasmid and (D) is the natural transformation with 100 ng of the pBWB162 plasmid. The graph (E) shows the efficiency comparison of 25 ng and 100 ng. On the y-axis is defined with the colony forming unit (CFU) and on the x-axis are the different conditions. Additionally, the p values between the two protocols and the amount of DNA was calculated.

To sum up, the natural transformation is successfull without any procession of the plasmid by an pBAV1k origin of replication. The efficiency is significantly higher with the higher amount of DNA (p value = 0,0879) and the highest comptence is in the exponential growth phase with the "log-phase" protocol which is congruent with the literature [5].

Natural transformation in co-culture

In the co-culture with A. baylyi ADP1, E. coli DH5α and Bacillus subtilis 168 (B. subtilis) was the amount of plasmid increased to 200 ng in our protocol to transform successful clones.

Figure 3. Natural transformation of A. baylyi ADP1 in a co-culture. (A) shows the counted colonies in a table. The co-culture natural transformation experiment was performed with 100 ng and 200 ng of the pBWB162 plasmid regarding the “overnight” protocol. (B+C) represents the natural transformation co-culture plates with 100 ng plasmid DNA in (B). The successful cloned colonies in (C) with 200 ng of plasmid DNA are depicted with white arrows.

Reference

[1] Development of a genetic toolset for the highly engineerable and metabolically versatile Acinetobacter baylyi ADP1. Biggs BW, Bedore SR, Arvay E, Huang S, Subramanian H, McIntyre EA, Duscent-Maitland CV, Neidle EL, Tyo KEJ. Nucleic Acids Res. 2020 Apr 4. pii: 5815822. doi: 10.1093/nar/gkaa167. 10.1093/nar/gkaa167 PubMed 32246719

[2] Leenhouts, K. J., Tolner, B., Bron, S., Kok, J., Venema, G., & Seegers, J. F. (1991). Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1. Plasmid, 26(1), 55–66. https://doi.org/10.1016/0147-619x(91)90036-v

[3] Biggs, B. W., Bedore, S. R., Arvay, E., Huang, S., Subramanian, H., McIntyre, E. A., Duscent-Maitland, C. V., Neidle, E. L., & Tyo, K. (2020). Development of a genetic toolset for the highly engineerable and metabolically versatile Acinetobacter baylyi ADP1. Nucleic acids research, 48(9), 5169–5182. https://doi.org/10.1093/nar/gkaa167

[4] Expression vectors for the engineering of genes and genomes in Acinetobacter baylyi ADP1. Murin CD, Segal K, Bryksin A, Matsumura I. Appl Environ Microbiol. 2011 Oct 21. 10.1128/AEM.05597-11 PubMed 22020504

[5] Hülter, N., Sørum, V., Borch-Pedersen, K., Liljegren, M. M., Utnes, A. L., Primicerio, R., Harms, K., & Johnsen, P. J. (2017). Costs and benefits of natural transformation in Acinetobacter baylyi. BMC microbiology, 17(1), 34. https://doi.org/10.1186/s12866-017-0953-2


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1364
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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