Difference: ProtocolsAcinetobacterBaylyiADP1 (1 vs. 2)

Revision 22022-03-18 - JeffreyBarrick

META TOPICPARENT	name="ProtocolList"

Acinetobacter baylyi ADP1 Overview

ADP1 Resources

Changed:

<
<

>
>

Genome Sequences
- ADP1 Wild-Type (GenBank: NC_005966.1)
  In general, use this genome as a reference when referring to coordinates of genes and features in the ADP1 genome.

Added:

>
>

- - BLAST a sequence against the ADP1 (WT) genome (at NCBI)
- ADP1 Barrick (GenBank: NC_005966.1)
  Incorporates a small number of mutations found in the WT ADP1 strain that the Barrick lab originally obtained from Valérie de Crécy-Lagard. Use for analyzing NGS sequencing results.
- [[][ADP1-ISx]]
  Strain created in the Barrick lab with all IS1236 elements deleted. (NEEDS LINK)

Changed:

<
<

| Quick Reference

>
>

Other Useful Sequences

Added:

>
>

- tdk-kanR cassette used for genome editing

Functional Genomics
- ADP1 pathways and gene functions (KEGG)
- ADP1 pathways and gene functions in (BioCyc)

Antibiotic Resistance

Added:

>
>

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 µg/ml	100 µg/ml	Resistant

Changed:

<
<

Spectinomycin

60 µg/ml

Sensitive

>
>

Kanamycin

12.5-6.25 µg/ml (3.125µg/mL doesn't inhibit)

50 µg/ml

Sensitive

MIC info from Barrick Lab results (G.S.)

Added:

>
>

Spectinomycin

60 µg/ml

Sensitive

Spontaneous resistance

Streptomycin

6.25-12.5 µg/ml (escapes observed at 6.25µg/mL)

25 µg/ml

Sensitive

Barrick Lab results (G.S.)

Deleted:

<
<

Kanamycin

12.5-6.25µg/ml (3.125µg/mL doesn't inhibit)

50 µg/ml

Sensitive

MIC info from Barrick Lab results (G.S.)

Chloramphenicol	4.0 µg/ml	20 µg/ml	Sensitive	Spontaneous resistance
Gentamycin	1.3 µg/ml	5 µg/ml	Sensitive
Tetracycline	1.0 µg/ml	10 µg/ml	Sensitive
Rifampicin	0.6 µg/ml	100 µg/ml	Sensitive	Spontaneous resistance

Changed:

<
<

Nalidixic acid (mutagen/causes DNA double strand breaks)

1.9µg/mL (no growth at 24hr, but growth at 48hr) (1µg/mL doesn't inhibit)

3.75 µg/ml

Sensitive

Barrick Lab results (G.S.)

>
>

Nalidixic acid (mutagen/causes DNA double strand breaks)

1.9 µg/ml (no growth at 24 hr, but growth at 48 hr) (1 µg/ml doesn't inhibit)

3.75 µg/ml

Sensitive

Barrick Lab results (G.S.)

Changed:

<
<

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

>
>

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

Changed:

<
<

Genome Resources: BLAST | BioCyc | Quick Reference

>
>

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.

Added:

>
>

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

META TOPICPARENT	name="ProtocolList"

Acinetobacter baylyi ADP1 Overview

ADP1 Resources

| Quick Reference

Antibiotic Resistance

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

View topic | History: r6 < r5 < r4 < r3 | More topic actions...