Acinetobacter baylyi ADP1 Overview

ADP1 Resources

  • Other Useful Sequences
    • pBTK622
      Plasmid containing the tdk-kanR cassette used for genome editing.

Working with ADP1

  • Culture conditions
    • LB is typically used when growing ADP1 in rich media.
    • Minimal Succinate (ABMS) media is used when growing ADP1 in minimal media.
    • Media can be supplemented with antibiotics for selection of strains transformed with resistance markers as described below.
    • ADP1 grows at both 30C and 37C. The Barrick Lab typically incubates ADP1 cultures at 30C with shaking at 200 rpm.

  • Storage
    • Cultures of ADP1 can be stored for a few days at 4C.
    • Glycerol or DMSO can be used as cryoprotectants when freezing cultures stocks (using the protocol here)

  • Genomic DNA extraction: The Barrick Lab uses a PureLink Genomic DNA Mini Kit for genomic DNA extractions. 30 minutes is sufficient incubation time at 55C for lysing cultures of ADP1.

  • Protocols: the following protocols are commonly used when working with ADP1
    • PCR Amplifying DNA.
    • Golden Transformation Creating a construct for integration into the ADP1 genome with Golden Gate ligation and transforming it into ADP1. Constructs can be used for gene deletion, inserting DNA sequences, or introducing mutations.
    • Growth curves Calculating growth rates using a plate reader.
    • Competence assay Calculating transformation rates and confirming competence of ADP1 mutants.

Antibiotic Resistance

ADP1 is intrinsically resistant to beta-lactam antibiotics. Do not use plasmids/constructs that have beta-lactamase (bla) as the selectable marker gene for ampicillin (or carbenicillin) resistance. Most other common antibiotic selection markers can be used.

We most commonly use kanamycin and spectinomycin resistance markers. The rate of spontaneous mutations to KanR is undetectable. SpecR mutants can arise at a low frequency (~10−8 to 10−9) due to chromosomal mutations, so only use this marker for transformations that have a significantly higher frequency than this.

Antibiotic MIC Standard Conc. Status Notes
Ampicillin (or carbenicillin) 128 g/ml 100 g/ml Resistant  
Kanamycin 12.5-6.25 g/ml (3.125g/mL doesn't inhibit) 50 g/ml Sensitive MIC info from Barrick Lab results (G.S.)
Spectinomycin ? 60 g/ml Sensitive Spontaneous resistance
Streptomycin 6.25-12.5 g/ml (escapes observed at 6.25g/mL) 25 g/ml Sensitive Barrick Lab results (G.S.)
Chloramphenicol 4.0 g/ml 20 g/ml Sensitive Spontaneous resistance
Gentamycin 1.3 g/ml 5 g/ml Sensitive  
Tetracycline 1.0 g/ml 10 g/ml Sensitive  
Rifampicin 0.6 g/ml 100 g/ml Sensitive Spontaneous resistance
Nalidixic acid (mutagen/causes DNA double strand breaks) 1.9 g/ml (no growth at 24 hr, but growth at 48 hr) (1 g/ml doesn't inhibit) 3.75 g/ml Sensitive Barrick Lab results (G.S.)

Source: Gomez MJ, Neyfakh AA. (2006) Genes involved in intrinsic antibiotic resistance of Acinetobacter baylyi. Antimicrob Agents Chemother 50:3562–3567. DOI: 10.1128/AAC.00579-06

Plasmids

Common high-copy number E. coli plasmids do not work in ADP1.

A synthetic plasmid (pBAV1K) and broad-host-range plasmid (RSF1010) have been used successfully. In our hands, pBAV1K is less reliable. It is also important to note that because plasmid DNA is linearized when taken up through the competence apparatus, that transformation of plasmids can be significantly less efficient than transformations used to incorporate DNA directly in the chromosome.

Origin Copy Number Example
ColE1 (pUC, pBR322, etc.) type plasmids Do not replicate NA
pBAV1K Low-medium copy number pBAV1K-T5-gfp
RSF1010 Low-medium copy number pBTK403
Edit | Attach | Watch | Print version | History: r6 < r5 < r4 < r3 < r2 | Backlinks | Raw View | More topic actions

 Barrick Lab  >  ProtocolList  >  ProtocolsAcinetobacterBaylyiADP1

Contributors to this topic Edit topic JeffreyBarrick, IsaacGifford
Topic revision: r6 - 2022-03-22 - 18:35:13 - Main.IsaacGifford
 
This site is powered by the TWiki collaboration platform Powered by Perl This site is powered by the TWiki collaboration platformCopyright ©2022 Barrick Lab contributing authors. Ideas, requests, problems? Send feedback