Barrick Lab :: ProtocolsAcinetobacterTransformation

---+ Transforming _Acinetobacter baylyi_ ADP1



---++ About _A. baylyi_ ADP1

_Acinetobacter baylyi_ ADP1 is a naturally competent bacteria with enormous potential for genome engineering due to the ease of genome manipulation its competence confers. ADP1 becomes competent after being diluted into fresh media from stationary phase and remains competent until late log phase. The following procedures are used for transforming a self-replicating plasmid into ADP1 or an integrating PCR product with homology to the ADP1 genome. Genomic DNA from marker carrying ADP1 strains can also be used as a transformation source. Discussions of plasmids that are functional in ADP1 and the procedure for genome modification will be provided elsewhere. 

---++ Transforming a plasmid or PCR product into ADP1
The following is a standard procedure for transforming plasmid or a PCR product into the ADP1 strain. 

   1 Grow a fresh culture of AB overnight in rich media. Generally this is done by inoculating a small amount of frozen glycerol stock or previous culture into 10mL of LB in a sterile 50mL flask and shaking in a 30°C incubator at 140RPM overnight. Alternatively, inoculate into 5mL of LB in a sterile test tube and shake at 30°C and 200RPM. 
   2 In a sterile test tube, combine 1mL fresh LB, >100ng of transforming DNA, and 70uL of overnight grown ADP1 culture. 
   3 Incubate the test tube in a shaking incubator for at least 3 hours, ideally overnight. 
   4 Plate dilutions of the transformed culture onto selective and non-selective media plates using sterile glass roller beads. Incubate the plates at 30°C overnight. 
   5 Pick colonies that grow on selective media and use PCR to assay for a successful transformation. Growth on non-selective but not selective plating suggests a failed transformation. 

---++ Notes/Considerations in ADP1 transformations
   * Growth and transformation occur with equal transformation frequency on appropriate minimal medias as in LB (examples: M9+1% glucose, S2 lactic acid media, and succinate minimal media - the current standard [[ProtocolsRecipesABMS]]). 
   * If using 100ng of PCR product with at least 1000bp flanking homology on either side of the transformed marker or ADP1 gDNA, you can expect transformation frequencies of around 10E-4.
   * Transformation frequency can be increased by increasing the concentration of transforming DNA, with a maximum transformation frequency (approx. 10E-3 to 10E-2) at 2ug/mL of ADP1 genomic DNA. 
   * Transformation frequency is sensitive to the amount of homology a construct has to the ADP1 genome for PCR products. 500bp on each flank should be used as a minimum for moderate efficiency (10E-5 transformation frequency at 100ng per 1mL transformation). A minimum of 250bp on each flank should be used in general as transformations with less homology are unreliable. 
   * Avoid transformations in the presence of chelating agents such as EDTA, as AB transformation has been found to be sensitive to divalent cation concentration. 
   * When plating ADP1, try to avoid the use wet plates or plates with aberrant surface texture. Wet plates will haze individual colonies and aberrant surface texture produces irregular morphologies. 
   * Linearized plasmid will not recircularize in an AB transformation unless two separate digestions with different cut sites on the plasmid are used. It is best to just transform whole plasmid. 
   * If plasmid transformation efficiency is too low with the above method, use a [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsAcinetobacterPuddleTransformation]['puddle transformation']]. This is performed by mixing 50uL of overnight grown culture with the transforming DNA of interest and spotting the 50uL onto an agar plate. If available, first lay a millipore membrane filter onto the plate and spot the culture+DNA mixture on to this, as it facilitates easier capture of transformants. After spotting the culture, let it grow in the 30°C incubator overnight. Scrape cells from the plate (or transfer the membrane if used) into 1mL sterile saline and plate dilutions of this onto selective media. Generally, one can assume the cells resuspended in 1mL of saline to have the same cell concentration as those grown overnight in LB. 

---++ Transformation efficiency / frequency
The following is a standard procedure for detecting transformation efficiency and  frequency in ADP1. 

   1 Grow a fresh culture of AB overnight in rich media. Generally this is done by inoculating a small amount of frozen glycerol stock or previous culture into 10mL of LB in a sterile 50mL flask and shaking in a 30°C incubator at 140RPM overnight. Alternatively, inoculate into 5mL of LB in a sterile test tube and shake at 30°C and 200RPM. 
   2 In the sterile test tubes, 1:100 dilute the overnight AB culture with LB and add 250 ng/ml DNA (as a standard amount for the comparison with previous results) to a final volume of 500µL.  
   3 Incubate the test tubes in a shaking incubator overnight (12-16 hr). 
   4 Plate 1:50 dilutions of the transformed culture onto selective plates (expect approximate 20 cells per plate), and properly dilution (normally E-5 to E-6 dilution) onto non-selective media plates using sterile glass roller beads. Incubate the plates at 30°C overnight. 
   5 Transformation frequency (TF) is Ns / Nnon, where Ns and Nnon are the resistant cell number and total cells number respectively. The transformation efficiency (TE) is the Ns / 250 with a unit of /ng.    

*Notes*
   * Experiment will have at least 6 replicates per strain per condition.
   * For each strain, the following controls should be set up: two controls for each strain with no DNA, one LB-DNA media control (no cells), and one blank media control.
   * TF and TE will vary with the strain condition, DNA sequence, length of DNA homology, and DNA concentration.


---++ Related references (links to articles)
*On natural competence in nature:* </br>
</br>
[[https://www.sciencedirect.com/science/article/pii/S096098221631003X][Natural competence for transformation]] - review mentions other naturally competent species </br>
[[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135910/][Pull in and Push Out: Mechanisms of Horizontal Gene Transfer in Bacteria]] - - as of 2018, this is the latest review on natural competence mechanisms. </br>
[[https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002394][Horizontal DNA Transfer Mechanisms of Bacteria as Weapons of Intragenomic Conflict]] - on the evolution of natural competence </br>
[[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701796/][Inter-species population dynamics enhance microbial horizontal gene transfer and spread of antibiotic resistance]] - how T6SS-mediated predation/killing by ADP1 increases cross-species HGT </br>
</br>
*On ADP1's natural competence:* </br>
</br>
[[https://www.nature.com/articles/ismej201535][Growth phase-specific evolutionary benefits of natural transformation in Acinetobacter baylyi]] </br>
[[https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-017-0953-2][Costs and benefits of natural transformation in Acinetobacter baylyi]] </br>
[[https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0182139][The role of core and accessory type IV pilus genes in natural transformation and twitching motility in the bacterium Acinetobacter baylyi]] </br>
[[https://www.ncbi.nlm.nih.gov/pubmed/11854504][Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-facilitated illegitimate recombination]] </br>
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Topic revision: r11 - 2020-02-14 - JeffreyBarrick