{"action":"opened","issue":{"url":"https://api.github.com/repos/dacarlin/robot-bagel/issues/3","repository_url":"https://api.github.com/repos/dacarlin/robot-bagel","labels_url":"https://api.github.com/repos/dacarlin/robot-bagel/issues/3/labels{/name}","comments_url":"https://api.github.com/repos/dacarlin/robot-bagel/issues/3/comments","events_url":"https://api.github.com/repos/dacarlin/robot-bagel/issues/3/events","html_url":"https://github.com/dacarlin/robot-bagel/issues/3","id":135160682,"number":3,"title":"TODO: Transformation module","user":{"login":"bmiles","id":3384543,"avatar_url":"https://avatars.githubusercontent.com/u/3384543?v=3","gravatar_id":"","url":"https://api.github.com/users/bmiles","html_url":"https://github.com/bmiles","followers_url":"https://api.github.com/users/bmiles/followers","following_url":"https://api.github.com/users/bmiles/following{/other_user}","gists_url":"https://api.github.com/users/bmiles/gists{/gist_id}","starred_url":"https://api.github.com/users/bmiles/starred{/owner}{/repo}","subscriptions_url":"https://api.github.com/users/bmiles/subscriptions","organizations_url":"https://api.github.com/users/bmiles/orgs","repos_url":"https://api.github.com/users/bmiles/repos","events_url":"https://api.github.com/users/bmiles/events{/privacy}","received_events_url":"https://api.github.com/users/bmiles/received_events","type":"User","site_admin":false},"labels":[],"state":"open","locked":false,"assignee":null,"milestone":null,"comments":0,"created_at":"2016-02-21T04:53:08Z","updated_at":"2016-02-21T04:53:08Z","closed_at":null,"body":"Create a module that describes transformation of BL21 (or other expression host) with mutant plasmid.\r\n\r\nSteps from https://github.com/dacarlin/bagel-protocol/blob/master/production.md:\r\n# Production and purification of BglB\r\n\r\nThis is a guide to the production and purification of β-glucosidase B from *P. polymxa* beginning with purified plasmid and ending with purified protein. This protocol and the associated scripts and data in this repository are part of the [Siegel group](https://sites.google.com/site/ucdsiegellab/)'s \"Bagel\" project. \r\n\r\nFor this procedure, typical yields are 1.0 ± 0.4 mg/mL for the wild type protein. \r\n\r\nBefore beginning, make sure you have the following ready and standing by:\r\n\r\n+ dry bath set to 42 C\r\n+ 37 C standing, 37 C shaking, and 18 C shaking incubators\r\n+ centrifuges for 50 mL Falcon tubes (up to 4,700 RPM) and 2 mL tubes (up to 14,700 RPM) \r\n\r\n## Day 0: Automated Kunkel mutagenesis \r\n\r\nAfter you have designed your set of mutants, an automated Kunkel mutagenesis procedure will be carried out on [Transcriptic](https://www.transcriptic.com/)'s workcell. The wild type Bagel plasmid is mutated to your designed sequence using the procedure that's described in [Thomas Kunkel's original procedure](http://www.pnas.org/content/82/2/488.full.pdf) and in [our group's manual Kunkel protocol](https://docs.google.com/a/ucdavis.edu/folderview?usp=sharing&id=0B3zIXvOOrmpqcEM5WWRadThsVUE). If you're interested, the [Python code that automates the Kunkel procedure and algorithmicly evaluates sequence verification](https://github.com/dacarlin/bagel-orders) is together but still a little messy (could use a second set of eyes on it).\r\n\r\nAfter your mutant plasmids have been Kunkel'd and sequenced, you'll get them in the mail ready for Day 1.\r\n\r\n### When you get your mutants \r\n\r\n**Note**: Everybody's mutants arrive together in a 96-well plate along with a CSV file that tells you where each mutant is on the plate. \r\n\r\nMaterial | Concentration  | Amount per reaction\r\n---------|----------------|--------------------\r\n2 mL tube | | 1\r\ntube label | | 1\r\n\r\n#### Mise en place\r\n\r\n*Mise en place* is a French culinary phrase which means \"putting in place\", as in \"set up\". It is used in professional kitchens to refer to organizing and arranging the ingredients that a cook will require for the items that are to be prepared during a shift ([Wikipedia](https://en.wikipedia.org/wiki/Mise_en_place)). \r\n\r\n+ waste bucket \r\n+ P200 and tips\r\n\r\n#### Instructions \r\n\r\n1. Find your mutant in the CSV file (make sure to verifiy the plate barcode matches the sheet)\r\n2. Write the mutant name on a ToughTag tube label and affix to a 2 mL tube \r\n2. Once you know which well your mutant is in, pipet the entire volume of that well into the tube\r\n3. Reseal the plate and store at -20 C\r\n3. Store your mutants at -20 C in the \"Bagel\" box or go immediately to Day 1\r\n\r\n## Day 1: Transformation \r\n\r\nIn this step, you will transform the plasmids encoding your mutant genes into *Escherichia coli BL21(DE3)*. Transformation is the process of physically shocking the bacteria to induce them to take up foreign DNA from the environment. Today, you will heat shock the *E. coli* to induce the uptake of the plasmid encoding the mutant you designed. \r\n\r\nMaterial | Concentration  | Amount per reaction\r\n---------|----------------|--------------------\r\nChemically-competent *Escherichia coli* BL21(DE3) | | 30 μL \r\nMutant plasmid solution | > 20 ng/μL | 2 μL\r\nTerrific broth (TB) | | 200 μL\r\nLB agar plates with kanamycin | | 1\r\nSterile glass beads | | 8–10 \r\n15 mL round-bottom culture tube | | 1\r\n\r\n### Mise en place\r\n\r\n+ waste bucket \r\n+ P2 and tips, P200 and tips\r\n+ Ice in ice bucket\r\n+ Pre-warmed 42 C dry bath \r\n\r\n### Hardware\r\n\r\n+ 37 C incubator\r\n\r\n### Instructions \r\n\r\n1. Set round-bottom tubes on ice, one per mutant\r\n2. When the tubes are ice cold, add plasmid and competent cells and incubate on ice for at least 10 minutes (20 min is better) \r\n3. Heat shock at 42 C for 90 seconds \r\n4. Incubate on ice for 90 seconds \r\n5. Add 200 µL Terrific Broth and recover with shaking at 37 C for 1 hour\r\n6. Place plates in incubator upside down to warm them\r\n7. Right before the hour is up, add glass beads to each plate\r\n6. Pipet 200 µL recovered cells on to plate and shake to spread out the cells\r\n7. After waiting a minute so that the liquid is absorbed, turn the plates upside down so that the beads fall onto the underside of the lid, and deposit beads into bleach solution for later recovery\r\n7. Incubate plate *upside down* overnight at 37 C\r\n\r\n### Tips and tricks \r\n\r\n+ Chemical competent cells should be kept on ice at all times and never warmed\r\n+ Program the thermocycler to perform steps 1-5\r\n+ Some tips for [troubleshooting transformations from New England Biolabs](https://www.neb.com/tools-and-resources/usage-guidelines/chemical-transformation-tips) \r\n+ If you are getting poor transformation efficiency, try recovering with 400 µL of media for 2 hours at 37 C\r\n\r\n## Day 2: Growth \r\n\r\nToday, you will record the number of colonies on your selection plate and start a culture from a single colony. \r\n\r\nMaterial | Amount per reaction\r\n---------|---------------------------\r\nTransformation plate | 1\r\nTerrific Broth | 5 mL\r\nKanamycin solution | 5 μL\r\nTube seal | 1\r\n\r\n### Mise en place \r\n\r\n+ 50 mL tube rack \r\n+ waste bucket \r\n+ pre-cut seals\r\n+ pipets and tips \r\n+ markers \r\n+ serological pipet aspirator \r\n+ 25 mL serological pipet\r\n\r\n### Instructions \r\n\r\n1. Record the number of colonies on your transformation plate \r\n2. Add kanamycin solution to Terrific Broth to make a batch of growth media for all of your mutants. Make enough for one extra culture\r\n3. Aliquot 5 mL growth media to a Falcon tube \r\n1. Using a pipet, carefully scrape a single colony from the plate and dip in the growth media in the Falcon tube, and pipet up and down a few times \r\n2. Repeat for the other plates \r\n5. Seal the cultures with tube seals \r\n6. Incubate with shaking at 37 C for 24 hours\r\n\r\n### Tips and tricks \r\n\r\n+ If you have a lot of colonies, divide the plate into quadrants, count one quadrant, and multiply the resultant number by 4 to estimate how many colonies you got\r\n"}}