(r4) ProtocolsAcinetobacterBaylyiADP1 < Lab

---+ _Acinetobacter baylyi_ ADP1 Overview

---++ ADP1 Resources

   *  Genome Sequences
      * [[https://github.com/barricklab/Abaylyi-EE/raw/master/reference/Acinetobacter-baylyi-ADP1-NCBI.gbk][ADP1 type strain (GenBank Format)]] %BR% In general, use this genome as a reference when referring to coordinates of genes and features in the ADP1 genome. We added the locations of IS<i>1236</i> elements to the linked sequence. Otherwise it is the same as the one on NCBI.
         *  [[https://www.ncbi.nlm.nih.gov/nuccore/NC_005966.1][Original sequence of the ADP1 type strain at NCBI (GenBank: NC_005966.1)]] 
         * [[http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&USER_FORMAT_DEFAULTS=on&PAGE=MegaBlast&PROGRAM=blastn&GAPCOSTS=0%200&MATCH_SCORES=1,-2&BLAST_SPEC=MicrobialGenomes_62977&EQ_MENU=Acinetobacter%20sp.%20ADP1%20%28taxid%3A62977%29&DATABASE=Complete_Genomes&BLAST_PROGRAMS=megaBlast&MAX_NUM_SEQ=100&SHORT_QUERY_ADJUST=on&EXPECT=10&WORD_SIZE=28&TEMPLATE_TYPE=0&TEMPLATE_LENGTH=0&FILTER=L&FILTER=m&DB_GROUP=AllMG&SUBGROUP_NAME=All_genomes&DB_SUBGROUP=Complete_Genomes&SHOW_OVERVIEW=on&SHOW_LINKOUT=on&ALIGNMENT_VIEW=Pairwise&MASK_CHAR=2&MASK_COLOR=1&GET_SEQUENCE=on&NUM_OVERVIEW=100&DESCRIPTIONS=100&ALIGNMENTS=100&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&OLD_BLAST=false][BLAST a sequence against this genome at NCBI]]
      * [[https://github.com/barricklab/Abaylyi-EE/raw/master/reference/Acinetobacter-baylyi-ADP1-WT.gff3][ADP1 Barrick Lab strain (GFF3 Format)]] %BR% Incorporates a small number of mutations found in the ADP1 strain that the Barrick lab originally obtained from Valérie de Crécy-Lagard. Use for analyzing NGS sequencing results.
      * [[https://github.com/barricklab/Abaylyi-EE/raw/master/reference/Acinetobacter-baylyi-ADP1-ISx.gff3][ADP1-ISx (GFF3 Format)]] %BR% Strain created in the Barrick lab with all IS<i>1236</i> elements deleted.

   * Other Useful Sequences
      * [[https://github.com/barricklab/Abaylyi-EE/raw/master/reference/pBTK622_tdk-kanR_cassette_for%20Golden_Transformation.gbk][pBTK622]] %BR% Plasmid containing the <i>tdk</i>-kanR cassette used for genome editing.
 
   * Functional Genomics
      * [[https://www.genome.jp/kegg-bin/show_organism?org=T00185][ADP1 pathways and gene functions (KEGG)]] 
      * [[http://biocyc.org/organism-summary?object=ASP62977][ADP1 pathways and gene functions in (BioCyc)]] 

---++ Working with ADP1

   *  Culture conditions</br>
      * 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 [[https://barricklab.org/twiki/bin/view/Lab/ProtocolsMediaRecipes][here]]).</br>
         * Media can be supplemented with antibiotics for selection of strains transformed with resistance markers as described below.
      * ADP1 grows at both 30°C and 37°C. The Barrick Lab typically incubates ADP1 cultures at 30°C with shaking at 200 rpm.

   *  Storage</br>
      * Cultures of ADP1 can be stored for a few days at 4°C.</br>
      * Glycerol or DMSO can be used as cryoprotectants when freezing cultures stocks (using the protocol [[https://barricklab.org/twiki/bin/view/Lab/ProtocolsFreezingStrains][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 55°C for lysing cultures of ADP1.

   *  Protocols: the following protocols are commonly used when working with ADP1
      * [[https://barricklab.org/twiki/bin/view/Lab/ProtocolsStandardPCR][PCR]] Amplifying DNA.</br>
      * [[https://barricklab.org/twiki/bin/view/Lab/ProtocolsAcinetobacterGoldenTransformation][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.</br>
      * [[https://barricklab.org/twiki/bin/view/Lab/ProtocolsGrowthRates][Growth curves]] Calculating growth rates using a plate reader.</br>
      * [[https://barricklab.org/twiki/bin/view/Lab/ProceduresCompetenceAssays][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<sup>&minus;8</sup> to 10<sup>&minus;9</sup>) 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 | %RED%Resistant%ENDCOLOR% | |
| 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.) |
| 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.) |
| 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) | 1.9 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.) |

*Source:* Gomez MJ, Neyfakh AA. (2006) Genes involved in intrinsic antibiotic resistance of _Acinetobacter baylyi_. _Antimicrob Agents Chemother_ *50*:3562&ndash;3567. [[https://doi.org/10.1128/AAC.00579-06][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 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 | %RED% Do not replicate %ENDCOLOR% | NA |
| pBAV1K | Low-medium copy number | [[https://www.addgene.org/26702/][pBAV1K-T5-gfp]] |
| RSF1010 | Low-medium copy number | [[https://www.addgene.org/110599/][pBTK403]] |
Barrick Lab > ProtocolList > ProtocolsAcinetobacterBaylyiADP1
Topic revision: r4 - 2022-03-18 - 19:04:44 - Main.IsaacGifford