# Easy E.coli Transformation for Amateur Biohackers and DIYbio
by [Cathal Garvey][], of www.indiebiotech.com

Creative Commons Attribution, Sharealike: Feel free to modify, share, or publish this work provided that credit is given to me for the original work (with a link to indiebiotech.com) and that the derived work or publication is released under the same license. Share, share alike.

## Design Considerations
The following protocol is designed to make it as easy for a beginner to make their first GMO bacterium as possible. With that in mind, it attempts to fulfil the following requirements:
* Non-shaking incubation
* Medium-volume liquid handling (no micropipettes)
* No expensive reagents (no DMSO)
* Optional centrifuge
* Optional autoclave/pressure cooker

## Minimum equipment
* Sterile Glass or Pasteur Pipettes or glass droppers, preferably graduated.
    - If using glass pipettes, you'll also need a pipette pump and a pipette brush, available cheaply on ebay.
    - Glass droppers must be sterilisable by pressure cooker.
    - Disposible pasteur pipettes should be graduated and sterile, pre-wrapped.
* 30C incubator - achievable with a pet terrarium thermostat and heat-mat in a polystyrene box.
* Crushed ice
* Microwave for sterilising media (pressure cooker preferred!)
* Sterile work area
    - Bunsen burner or blowtorch-facing-up, or
    - Jury-rigged glove-box with inner surface sterilised, or
    - A good HEPA-rated air purifier with a fresh filter pointing at work area.
* Lab strain, plasmid-free E.coli
* Desired DNA plasmid
* Ingredients for basic broth:
    - Soy Protein (or lactose-free casein)
    - Yeast Extract with no added salt (or, balance salt content against your added salt below)
    - Salt, non-iodised, preferably without preservatives
    - Bromelain or Papain digestive tablets, or Meat tenderiser
* Additional ingredients for TSS-broth:
    - PEG-3350, sold under a number of brand names as a laxative: Miralax, GoLytely, GlycoLax, etc. Probably available through pharmacy over the counter, or internet.
    - MgSO4, sold as “Epsom Salt” in pharmacies as a bath salt.
* Fine Sieve, Coffee filters, funnel, cotton wool to clarify broth
* Glassware
    - Preferably actual lab glassware, available from brouwland.com or various ebay sellers. Alternatively;
    - Recycled jam jars for “Flasks”
    - Smaller jars for “test tubes”
    - Wide jars or glass ramekins for “Petri Dishes”.

## Transforming E.coli by a modified TSS method:
1. Prepare nutrient broth for E.coli (1), and set some aside to make TSS-broth (2).
2. Sterilise both in a microwave (3).
3. Under sterile conditions, place enough growth broth in a sterile glass container so that it is 3-5mm deep.
4. Under sterile conditions (4), inoculate the shallow broth with some lab-strain, plasmid-free E.coli.
5. Incubate the cells for 3-5 hours at 30C or 37C, until they start to look cloudy, swirling them occasionally (every 30mins or hour).
6. If you have a centrifuge, then centrifuge 2mls of the cells gently (less than 2000g, bottom setting on a dremelfuge) to separate them from the broth, then resuspend them in 0.2mls of TSS-broth.
7. If you have no centrifuge, take 0.2mls of cells and mix them with 0.2mls of 2x concentrated TSS-broth.
8. Leave the cells chill on ice for 10 minutes.
9. Add 0.05ml of plasmid DNA solution. If you have no micropipette, you can use a sterile plastic inoculating loop to transfer a loopful of DNA solution.
10. Flick or stir cells gently to mix. Then leave on ice for 30 minutes.
11. Add 0.5mls of TSS-broth, and incubate the cells for 1hr at 30C, or 30 minutes at 37C.
12. Flick-mix the cells and then streak or spread 0.1-0.5ml of cell suspension on normal (non-TSS) antibiotic-selection agar. It helps if you had allowed the agar plate to dry a little bit after pouring, so the cell mix can soak in.
13. _Invert_ the petri dishes so the agar surface is pointing down. Place them facing this way into the incubator and let them grow overnight. Check for colonies tomorrow. If you've used GFP or Lux, and any necessary gene-inducers are there to switch on the genes, transformed colonies should be fluorescent or glow-in-the-dark, respectively.
14. Pick transformed colonies and grow them separately in their own antibiotic-broth. If you have not used an obvious reporter such as GFP or Lux, you should pick ten colonies, and perform a miniprep or other DNA extraction on them so you can verify by electrophoresis that they have the intended DNA. Otherwise, expect that some of the colonies you see are actually random mutants.
15. If you isolate a successfully transformed cell, store at 4C by stabbing into a tube full of fresh, antibiotic-supplemented nutrient agar. At 4C, a “stab culture” can survive stably for a long time. Don't keep in a food-fridge; it's E.coli!

## (1) Preparing Nutrient Broth for E.coli:
1. In 500mls water, mix 5g salt-free Yeast Extract, 2.5g non-iodised table salt, 5g casein or soy protein isolate, one crushed bromelain digestive aid tablets or a half-teaspoon of meat tenderiser.
2. Mix well, breaking up air-pockets and dissolving the yeast extract and salt. Dissolve as much of the protein as possible. Leave somewhere warm for 2hrs for the tenderiser/bromelain to act on the soy protein/casein.
3. Heat until steaming, stirring well, then filter; first through a fine sieve, then through a coffee filter, then through two fresh coffee filters. Keep filtering until transparent. As a final, slow filter, you can gently pack some cotton wool into a funnel, and filter through the packed cotton wool.
4. When fully filtered, divide into 50ml or 100ml volumes, add 20g/L Agar if Agar is desired, and sterilise in the microwave (detailed in protocol 3a, below) or pressure cooker (3b). Remember not to seal lids when sterilising!
5. When sterilised, allow to cool, add appropriate amount of antibiotics to some, and then leave in the fridge until use.

## (2) Preparing TSS-Broth for E.coli:
1. Prepare broth as detailed in (1) above, until step 4. Follow step 2 below before microwave-sterilising.
2. Additives to normal broth for TSS-broth:
1. To make 100mls non-concentrated TSS-broth, add the following to 50mls of broth:
1. PEG-3350 powder, 10g
2. MgSO4, 0.55g
3. Extra broth to bring the volume to 100mls.
2. To make 100mls 2x-concentrated TSS-broth, add the following to 50mls of broth:
1. PEG-3350 powder, 20g
2. MgSO4, 1.1g
3. Extra broth to bring the volume to 100mls.
3. Stir or swirl until entirely dissolved. Warm in microwave to aid dissolution if necessary.
4. Now proceed to microwave or pressure-cooker sterilisation as detailed in (3) below.

## (3a) Sterilising in a Microwave
The below assumes you are using a rotating turntable microwave rated at 1kw. You may have to adjust the times for weaker or more powerful microwaves.

For liquid samples in glass vessels, place the lid on the vessel but do not seal the lid! Explosions may result if the lid is sealed! Only seal after sterilising.

Furthermore, only sterilise one thing at a time. Hotspots and coldspots mean that multiple things will experience different levels of heat transfer. Place the single item in the middle of the microwave, where it has probably been tuned to direct the most heat.

For a 25ml sample, sterilise by microwaving for 5-7 minutes.
For a 50ml sample, sterilise by microwaving for 7-9 minutes.
For a 100ml sample, sterilise by microwaving for 9-11 minutes. 

You can use this method to kill bacteria after an experiment as well, and you should do so.

## (3b) Sterilising in a Pressure Cooker
If you have a pressure cooker, _and know how to use it_, you can alternatively (and, it is thought, more reliably) sterilise things by cooking them at full pressure for 20 minutes. That is, bring the cooker to full pressure, then start counting 20 minutes, then let it cool on its own. Lids should still be loose on vessels during sterilisation in a pressure cooker. Seal them afterwards.

You can use this method to kill bacteria after an experiment as well, and you should do so.

## (4) Sterile Working Conditions
To keep wild bacteria out of your experiments, you have a few options:
A blue-flamed bunsen burner or blowtorch can be directed upwards, creating a strong updraft that helps to keep dust from settling on experiments. Within about 10cm of a blue, upward flame, work is pretty sterile.
Working under a good, high-rating HEPA air purifier's air-flow is another useful way to get sterile conditions.
You can also make a sterile area using a plastic box with holes cut in the side for your hands. Sterilise the inside of the box with bleach or alcohol spray, do the same with the surface underneath, then invert the box and block the arm-holes until you use the area. Sterilise the surfaces of everything you bring into the box with alcohol or disinfectant spray first. Wear rubber or latex gloves, and wash your hands and forearms before and after work. Sterilise the gloves with disinfectant or alcohol spray.
When working, keep lids on until you need to get access to a broth or bacteria, then cover again immediately.
Have an empty jar or two that you can use to dispose of used pipettes, toothpicks, loops or other tools.

## (5) Sterilising Glassware in an Oven
1. To sterilise glass items before use, wrap lid-less items such as pipettes in aluminium foil, and place metal lids on test tubes or jars but do not tighten. If test tubes have plastic lids, remove these and wrap the test tubes in foil; sterilise lids separately in a pressure cooker.
2. Place the items in a fan-assisted oven, and close the door. Place some tape on the door to gently seal it shut, so someone does not open it mid-cycle; the glass may shatter if the oven is opened during this process.
3. Set the oven to 200C and let it come up to heat.
4. Once at 200C, set a timer for 1:30 hours.
5. After 1:30 hours, turn off the oven and allow it to cool to room temperature.
6. Remove glassware carefully. Seal jar lids, leave other items in foil wrapping.

## License
This work is licensed under a [Creative Commons BY-SA 3.0][] license.

## References:
### Academic
The TSS method was adapted from the following source: 
* “One-step Preparation of Competent Escherichia coli: Transformation and Storage of Bacterial Cells in the Same Solution", Chung, C.T. and Niemela, S.L. and Miller, R.H., Proceedings of the National Academy of Sciences, 1989. Available here: http://www.pnas.org/content/86/7/2172.full.pdf+html

[Creative Commons BY-SA 3.0]: https://creativecommons.org/licenses/by-sa/3.0/
[Cathal Garvey]: http://biohacking.cathalgarvey.me