(r3) ProtocolsP1Transduction < Lab

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---+ P1 Transduction in _Eschericha coli_

This protocol is adapted from Thomason, L. C., Costantino, N. and Court, D. L. 2007. _E. coli_ Genome Manipulation by P1 Transduction. Current Protocols in Molecular Biology. 79:V:1.17:1.17.1&ndash;1.17.8. (DOI: 10.1002/0471142727.mb0117s79)

*Always use filter tips when pipetting phage lysates.

---++ Choosing P1 donor strain for deconvoluter strains

   1 Download Excel book attached to this page titled "Deconvoluter-well-lookup-table.xlsx".
   1 Enter genomic location of mutation to be replaced in cell I4 (highlighted yellow next to top green arrow). 
      * Genomic locations are to be relative to MG1655 background of the donor strains.
      * No other cell may be edited by default as the workbook is locked.
      * If workbook not behaving correctly or you believe there is an error in calculations inform Dan.
   1 The strain with the closest Kan marker will appear in cell K11 (highlighted green with bold text next to bottom green arrow).
   1 In the event of a tie the nearest 2 strains will be listed in K11 and K12.
   1 Column F will display a heat-map of the distance to all strains with green cells being the closest. 
      * No experimental evidence known for nearest strain not being optimal <strong>*BUT*</strong> could choose "next best" based on this column.
   1 Lysate for all 94 deconvoluter donor strains has been prepared and are stored in the deli fridge in B lab.

---++ Preparation of P1 Liquid Lysate
   1 Inoculate a 5ml overnight culture of donor strain in LB. 
      * If you'd like to make a lysate and do a transduction on the same day, inoculate the recipient strain along with the donor strain.
   1 Dilute saturated culture 1:100 into 5ml LB + 50ul of 20% glucose (final concentration = 0.2%) and 25 ul of 1M CaCl<sub>2</sub> (final concentration = 5mM).
   1 Incubate 30 to 45 minutes at 37°C with shaking. Before proceeding to the next step, visually inspect the culture for cells. The culture should be slightly cloudy and you should see swirls when it is held up to the light.
   1 Add 100 µl of P1 phage stock. 
      * Some notes on phage titer: A good titer for a phage stock is 10<sup>9</sup> to 10<sup>10</sup> pfu/ml. If the transduction fails, consider determining the titer of your phage stock.
   1 Continue to incubate the culture, shaking at 37°C, until the culture lyses. This typically takes ~3 hours. 
      * The culture should be clear and may contain clumps of floating debris. An unlysed culture will look smooth and silky.
   1 Add 200µl of CHCl<sub>3</sub> and continue shaking for 5 minutes. 
      * The CHCl<sub>3</sub> ensures complete cell lyses and kills the bacterial donor strain. CHCl<sub>3</sub> will not harm the phage, however it should be removed before storing at 4°C because it can lead to decreased viability of the stock.
   1 Transfer the supernatant to a 15ml conical tube and centrifuge for 5 mins at ~9200 x g, 4°C.
   1 Transfer ~4-5mL of the supernatant (your P1 lysate) into new 15mL conicals. 
      * Take care to not disturb the CHCl<sub>3</sub> or any debris which may have pelleted.
      * Phase lysates can be stored at 4°C for several years.
      * Additionally, the lysate can be further purified using a 0.45µm filter to remove any residual bacterial cells. This can also be done at any time later. In particular consider doing this if plating 100µl of lysate not exposed to cells yields colonies.

---++ Perform P1 Transduction

   1 Inoculate a 5ml overnight culture of the recipient strain in LB. 
      * Again, this step can be started at the same time donor cultures are started if you wish to do transduction on same day lysate generated.
   1 Pellet 1.5ml of saturated culture, 2 min at maximum speed.
   1 Resuspend the cells in 0.75ml in sterile P1 salts solution.
   1 Mix 100ul of cells + P1 salts mixture with 1-10ul of P1 lysate. 
      * As a negative control, include a tube containing 100ul of cells without lysate.
      * If using the lysate for the first time or troubleshooting a transduction, try varying the amount of lysate that is added to the cells - ex: up to 200µl. 
         * To date, 1-5µl has proven sufficient for P1 Transductions using the deconvoluter donor strains.
   1 Allow the phage to adsorb to the cells for 30 min at 37°C.
   1 Add 1ml of LB + 200ul of 1M sodium citrate. 
      * The sodium citrate will minimize secondary infection by chelating the calcium, which is necessary for phage to adsorb to bacteria.
   1 Incubate for 1 hour at 37°C with shaking.
   1 Harvest the cells by centrifuging for 2 mins at maximum speed. Remove and discard the supernatant.
   1 Resuspend the cells in 100ul of LB and spread on selective transduction plates containing 5mM sodium citrate. 
      * Spread 100ul of P1 phage on another selective plate as a negative control if the lysate has not been tested previously, or there is concern it has become contaminated with bacteria.
      * It is good practice to supplement selective plates with 5mM sodium citrate to prevent reinfection of any residual phage into the recipient strain. Additionally, you may have to adjust the antibiotic concentration.
   1 Incubate plates overnight.
   1 Pick colonies from plates and re-streak on selective plates, incubate overnight. 
      * Cell-only plates should have no colonies.
      * If using multiple concentrations of P1, pick colonies from the plate with the least amount of phage.
      * Usually plates with the lowest concentration of phage will have the fewest colonies.
      * If colonies are present on the phage-only plates, either the selection did not work or the phage lysate is contaminated with bacteria.
   1 Confirm the genotype by PCR. It is also a good idea to sequence upstream/downstream of the insertion site, especially when the donor and the recipient are different _E.coli_ strains.

---++ Preparation of stock solutions
| Solution | Preparation |
| P1 Salt solution | To be entered |
---++ Positive controls

 Specifics need to be listed, but MG1655 cells should serve as P1 transduction positive control for deconvoluter experiments.

-- Main.DaciaLeon - 25 Aug 201 6

-- Main.DanielDeatherage - 23 Feb 2017 updates for deconvoluter table and experiences
Attachments
Topic attachments
I	Attachment	History	Action	Size	Date	Who	Comment
xlsx	Deconvoluter-well-lookup-table.xlsx	r1	manage	50.4 K	2016-10-26 - 15:21	DanielDeatherage	Excel book to determine optimal deconvoluter strain to use for p1 transduction
Topic revision: r3 - 2017-02-23 - DanielDeatherage