Difference: ProtocolsAcinetobacterBaylyiADP1 (1 vs. 6)

Revision 62022-03-22 - IsaacGifford

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Acinetobacter baylyi ADP1 Overview

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

  • Functional Genomics
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Working with ADP1

  • Culture conditions
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    • LB is typically used when growing ADP1 in rich media and Minimal Succinate (ABMS) media is used when growing ADP1 in minimal media (recipes for both medias can be found here).
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    • 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.

Revision 52022-03-22 - JeffreyBarrick

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Acinetobacter baylyi ADP1 Overview

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

Revision 42022-03-18 - IsaacGifford

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Acinetobacter baylyi ADP1 Overview

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Working with ADP1

  • Culture conditions
    • LB is typically used when growing ADP1 in rich media and Minimal Succinate (ABMS) media is used when growing ADP1 in minimal media (recipes for both medias can be found here).
      • 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.

Revision 32022-03-18 - JeffreyBarrick

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Acinetobacter baylyi ADP1 Overview

ADP1 Resources

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

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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 linearizes 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.
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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 linearizes 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

Revision 22022-03-18 - JeffreyBarrick

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Acinetobacter baylyi ADP1 Overview

ADP1 Resources

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| Quick Reference

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Antibiotic Resistance

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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  
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Spectinomycin ? 60 g/ml Sensitive  
Streptomycin 6.25-12.5 g/ml (escapes observed at 6.25g/mL) 25 g/ml Sensitive Barrick Lab results (G.S.)
 
Kanamycin 12.5-6.25g/ml (3.125g/mL doesn't inhibit) 50 g/ml Sensitive MIC info from Barrick Lab results (G.S.)
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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
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Nalidixic acid (mutagen/causes DNA double strand breaks) 1.9g/mL (no growth at 24hr, but growth at 48hr) (1g/mL doesn't inhibit) 3.75 g/ml Sensitive Barrick Lab results (G.S.)
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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.)
 
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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
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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

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Genome Resources: BLAST | BioCyc | Quick Reference
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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 linearizes 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

Revision 12022-03-17 - JeffreyBarrick

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META TOPICPARENT name="ProtocolList"

Acinetobacter baylyi ADP1 Overview

ADP1 Resources

| Quick Reference

Antibiotic Resistance

Antibiotic MIC Standard Conc. Status Notes
Ampicillin (or carbenicillin) 128 g/ml 100 g/ml Resistant  
Spectinomycin ? 60 g/ml Sensitive  
Streptomycin 6.25-12.5 g/ml (escapes observed at 6.25g/mL) 25 g/ml Sensitive Barrick Lab results (G.S.)
Kanamycin 12.5-6.25g/ml (3.125g/mL doesn't inhibit) 50 g/ml Sensitive MIC info from 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) 1.9g/mL (no growth at 24hr, but growth at 48hr) (1g/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

Genome Resources: BLAST | BioCyc | Quick Reference

 
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