iGEM 2015 — Team Purdue

# Team:Purdue/Notebook

![Team Banner](https://static.igem.org/mediawiki/2015/8/8b/Team_Purdue_banner.jpg)

[* INTRODUCTION](https://2015.igem.org/Team%3APurdue)
* BACKGROUND
  [+ The Problem](https://2015.igem.org/Team%3APurdue/TheProblem)
  [+ Organisms](https://2015.igem.org/Team%3APurdue/Organisms)
  [+ Enzymes](https://2015.igem.org/Team%3A%20Purdue/Enzymes)
* PROJECT
  [+ Description](https://2015.igem.org/Team%3APurdue/Description)
  [+ Project Development](https://2015.igem.org/Team%3APurdue/ProjectDevelopment)
  [+ Our Solution](https://2015.igem.org/Team%3APurdue/OurSolution)
* PARTS
  [+ Team Parts](https://2015.igem.org/Team%3APurdue/Parts)
  [+ Basic Parts](https://2015.igem.org/Team%3APurdue/Basic_Part)
  [+ Composite Parts](https://2015.igem.org/Team%3APurdue/Composite_Part)
  [+ Part Collection](https://2015.igem.org/Team%3APurdue/Part_Collection)
  [+ Parts Used](https://2015.igem.org/Team%3APurdue/PartsUsed)
  [+ Parts Submitted](https://2015.igem.org/Team%3APurdue/PartsSubmitted)
[* NOTEBOOK](https://2015.igem.org/Team%3APurdue/Notebook)
[* ATTRIBUTIONS](https://2015.igem.org/Team%3APurdue/Attributions)
[* OUR PROCESS](https://2015.igem.org/Team%3APurdue/Collaborations)
[* HUMAN PRACTICES](https://2015.igem.org/Team%3APurdue/Practices)
[* SAFETY](https://2015.igem.org/Team%3APurdue/Safety)
[* MODELING](https://2015.igem.org/Team%3APurdue/Modeling)

# Notebook

## Protocols

## Yeast Transformation

Written up by Bowman Clark

#### Purpose

This is a protocol for the transformation of plasmids into yeast.

#### Materials

* Yeast culture
* 1.5 mL sterile tube
* Pipette
* One-Step buffer
* 10 microliters DTT - 1g - $42.90 Sigma Aldrich
* 5 microliters ssDNA - 1mg - $32 Sigma Aldrich
* 3 microliters plasmid
* Sterile water

One-Step buffer - adding 80 microliters of (Possibly divide by factor of 10)

* 2.06g LiAc - 250 g = $51.50
* 40g PEG
* Make 80 mL solution in water

#### Equipment - 8000 rpm microcentrifuge – 134 - 42°C incubator - Water Bath B025 - vortex – 134 Protocol 1. Start a 5 mL yeast culture and grow overnight 2. Take 500 μl of overnight culture and place in 1.5 mL sterile tube. Label tube with strain name and plasmid name 3. Spin in microcentrifuge for 30 sec at 8K rpm to pellet cells 4. Remove and discard supernatant 5. Resuspend pellet in 1 mL sterile water by gently pipetting up and down 6. Pellet the cells and centrifuge at 8000 rpm for 30 sec again 7. Remove and discard supernatant 8. Heat ssDNA at 90-100°C for 5 min 9. Add 80 μl One-Step buffer (vortex after each addition), 1 μl of 1M DTT, 5 μl of ssDNA, and 5 μl of plasmid. Mix well 10. Incubate at 42°C for 30-60 min 11. Add 1 mL sterile water to transformation, mix and spin at 8000 rpm for 30 seconds 12. Remove 800 μl liquid from tube and resuspend transformed cells in residual liquid 13. Plate on selective media 14. Parafilm plates and allow to grow for 2-4 days Discussion 1. Add more plasmid if transformations are not working 2. Make a new 1-step buffer for every transformation - DO NOT USE AN OLD STOCK Source Nina Serratore from the Briggs Lab Others: Clontech Yeast Protocol Handbook, [http://www.bio-protocol.org/e96](%E2%80%9Chttp%3A//www.bio-protocol.org/e96%E2%80%9D) Kiran Aslam, Hansen 235 (Hazbun Lab) Paragraph form Inoculate 5 mL of yeast broth and grow the culture up overnight. Pipette 500 μL of the culture into a sterilized 1.5 mL tube. Label the tube. Centrifuge the tube for 30 seconds at 8000 rpm to pellet the cells. Discard the supernatant. Add 1mL of sterile water to the tube and resuspend the cells by gently pipetting up and down. Centrifuge the tube again for 30 seconds at 8000 rpm to pellet the cells. Discard the supernatant. Add the following and vortex after each addition: 80 μL One-step buffer, 10 μL of 1 M DTT, 5 μL of ssDNA, and 3 μL of plasmid. Mix well. Incubate the tube at 42 degrees C for 30 minutes. Add 1 mL of sterile water to the transformation and mix. Centrifuge again at 8000 rpm for 30 seconds. Remove and discard 800 μL of liquid from the tube, and resuspend the cells in the remaining liquid. Plate on selective media. Ligation Protocol written by Bowman Clark Purpose Protocol for preparing Parts for assembly Materials - 21 ul of digested plasmid - 15 ul of each digested part - 24 ul ddH20 - 9 ul 10x T4 Ligase Buffer - 6 ul T4 Ligase - PCR Tubes Equipment - Thermocycler (BIND 222) - Water Bath (BIND 134) Protocol --- 1. Add appropriate amounts of vector, insert, and water to PCR tubes according to the table below 2. Heat tubes for 2 minutes in water bath at 42 C to free up sticky ends 3. Pipette 10x buffer up and down and add 1.5 ul to each tube 4. Pipette T4 ligase up and down and add 1 ul to each tube, pipetting up and down in each tube after the addition of the T4 5. Incubate at 16 C for 16 hours in thermocycler Ligation Table | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | | Item: | 1:06 | 1:03 | 1:01 | 3:01 | 6:01 | 1:00 | | Vector (ul) | .7 | 1.4 | 3 | 6 | 6 | 3 | | Insert Total (ul) | 8.4 | 8.4 | 6 | 4 | 2 | 0 | | dH2O (ul) | 2.4 | 1.7 | 3.5 | 2.5 | 4.5 | 9.5 | | 10x Buffer (ul) | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | | T4 Ligase (ul) | 1 | 1 | 1 | 1 | 1 | 1 | | Total (ul) | 15 | 15 | 15 | 15 | 15 | 15 | Discussion Video explanation: [https://vimeo.com/45842813](%E2%80%9Chttps%3A//vimeo.com/45842813%E2%80%9D) Source iGEM Protocol: [http://parts.igem.org/Help:Protocols/Ligation](%E2%80%9Chttp%3A//parts.igem.org/Help%3AProtocols/Ligation%E2%80%9D) Protocol Paragraph Form: --- First, add the appropriate amount of vector, insert and water to PCR tubes for desired ligation ratios. Next, heat the tubes in a water bath at 42 C to free up the sticky ends. Pipette the T4 Ligase Buffer and add 1.5 ul toT4 each tube. Then pipette the T4 Ligase up and down and add 1 ul to each tube pipetting the mixture up and down after addition. Finally incubate the tubes at 16 C for 16 hours in the thermocycler. Restriction Digest Protocol Written up by Bowman Clark (Adjusted by Lexi Petrucciani) Purpose To prepare parts for assembly Materials - Ice and bucket/container - Eppendorf Tubes - Part A (Purified DNA, > 16ng/ul) - Part B (Purified DNA, > 16ng/ul) - Linearized plasmid backbone (25ng/ul) - dH2O - NEB Buffer 2 - BSA - Restriction Enzymes: EcoRI, SpeI, XbaI, PstI, DpnI - Thermal cycler or Thermomixer Equipment List all the equipment needed to complete this protocol, including what lab # in Bindley or another building it can be found in Protocol 1. Keep all enzymes and buffers used on ice. 2. Thaw NEB Buffer 2 and BSA in room temperature water. Mix by shaking the tubes, and flick/spin them to collect the liquid at the bottom of the tube. 3. Add 500ng of DNA to the appropriately labelled tube. Add distilled water to the tubes for a total volume of 36.5ul in each tube. 1. Calculation example (with 25ng/ul as DNA sample concentration): 500ng ÷ 25ng/ul = 20ul of DNA sample 36.5ul (total volume) – 20ul (DNA sample) = 16.5ul of distilled water 4. Pipet 5ul of NEB Buffer 2 to each tube. 5. Pipet 0.5ul of BSA to each tube. 6. Add 4uL of the appropriate enzymes 1. For 3A: 1. Part 1: EcoRI and SpeI 2. Part 2: XbaI and PstI 3. Backbone: EcoRI and PstI(IF NOT ALREADY LINEARIZED) 2. For Standard: 1. Insert: EcoRI and SpeI 2. Vector: EcoRI and XbaI 7. The total volume in each tube should be 50ul. Mix well by vortexing. Spin the samples briefly to collect all of the mixture to the bottom of the tube. 8. Incubate the restriction digests at 37°C for 4 hours, then use immediately or store at -20C. 3A Assembly | | | | | | --- | --- | --- | --- | | Item: | Part A: | Part B: | Backbone: | | DNA | 500ng | 500ng | 500ng | | dH2O | Adjust to 36.5ul | Adjust to 36.5ul | Adjust to 36.5ul | | NEB Buffer 2 | 5ul | 5ul | 5ul | | BSA | .5ul | .5ul | .5ul | | Enzyme 1 | 4ul EcoRI | 4ul XbaI | 4ul EcoRI | | Enzyme 2 | 4ul SpeI | 4ul PstI | 4ul PstI | Discussion IF THE BACKBONE IS ALREADY LINEARIZED THEN YOU DON’T NEED TO DIGEST IT. Video explanation: [https://vimeo.com/45793760](%E2%80%9Chttps%3A//vimeo.com/45793760%E2%80%9D) Source iGEM Protocol [http://parts.igem.org/Help:Protocols/Restriction\_Digest](%E2%80%9Chttp%3A//parts.igem.org/Help%3AProtocols/Restriction_Digest%E2%80%9D) Protocol Paragraph Form 3A Digest Add 500 ng of DNA to an Eppendorf Tube. Add dH20 to 36.5 ul. Add 5ul NEB Buffer 2, 0.5 ul BSA, and 4 ul of each enzyme. Vortex, then incubate at 37C for 4 hours. Experiment Log | | | | | --- | --- | --- | | Experiment: | Name(s) | Date | | Miniprep of yeast backbones from E. coli culture (pRS313, pRS315, pRS316) | Everyone (and the high schoolers) | 6/15/2015 || Preparation of competent E. coli | Mark, Lexi | 6/19/2015 | | Transformation of backbones into yeast culture | Tony, Bo | 6/22/2015 | | 3A Assembly of killswitch promoter to RBS | Everyone | 6/23/2015 | | Transformation of GFP into E. coli to test competency of cells we made ourselves | Bo, Lexi | 6/24/2015 | | Digestion of promoter, rbs, lysing agent, and terminator | Bo, Lexi, Kate | 6/24/2015 | | Ligation and transformation of promoter rbs lysing agent and terminator | Bo, Lexi, Kate | 6/25/2015 | | PCR of G-Block Fragments | Mark, Melissa, Erich, Suraj, Jill | 6/25/2015 | | | | //2015 | | Resuspension and Dilution of G-Blocks and Primers | Mark, Melissa, Erich, Suraj, Jill | 6/25/2015 | | PCR (3 replicates) of LiP1 and LiP2 | Mark, Melissa, Erich, Suraj, Jill | 6/26/2015 | | PCR (3 replicates) of Lac1 and Lac2 | Mark, Melissa, Erich, Jill | 6/26/2015 | | Run a gel to check digestion of promoter rbs lysing agent and terminator | Bo, Lexi, Kate | 6/26/2015 | | PCR (3 replicates) of MnP1 and MnP2 | Mark, Melissa, Jill | 6/29/2015 | | PCR (3 replicates) of AKR1, VP1, VP2 | Melissa | 6/30/2015 | | PCR (3 replicates) of AKR2, Tyr1, Tyr2 | Melissa, Erich | 7/1/2015 | | Gel of Lac1, Lac2, MnP1, MnP2, VP1 | Mark, Jill | 7/1/2015 | | PCR (3 replicates) of all gBlocks | Mark, Melissa, Jill, Erich | 7/1/2015 | | Miniprep of promoter, rbs, lysing agent, and terminator | Bo, Lexi, Kate | 7/6/2015 | | Digestion of promoter with rbs and lysing agent, with terminator | Bo, Lexi, Kate | 7/7/2015 | | PCR Purification (of MnP1&2, AKR1&2, VP1&2, and Tyr1) | Suraj, Melissa | 7/7/2015 | | PCR of all G-Block Fragments | Jill, Mark, Erich | 7/7/2015 | | Inoculation of liquid yeast cultures | Mark | 7/7/2015 | | LB Agar with Amp plates | Bo, Lexi, Kate | 7/8/2015 | | Gel of LiP1, LiP2, Lac1, Lac2, Tyr2 | Mark, Melissa, Erich, Suraj, Jill | 7/8/2015 | | Gibson Assembly with PCR purified gBlocks (Mn1+Mn2, AKR1+AKR2, VP1+VP2) | Mark | 7/8/2015 | | Gel of LiP1, LiP2, Lac1, Lac2, Tyr2 (of second PCR attempt) | Mark, Melissa, Erich, Suraj, Jill | 7/8/2015 | | PCR Purification of LiP1 and LiP2 | Jill, Erich, Melissa, Suraj | 7/8/2015 | | Gibson Assembly with PCR purified gBlocks (LiP1 + LiP2) | Jill, Erich, Melissa, Suraj | 7/8/2015 | | E. Coli Transformation of Promoter-RBS and LysingAgent-Terminator Ligations | Bo, Lexi, Kate | 7/8/2015 | | Yeast Transformation of backbones pRS313, pRS315, and pRS316 | Mark, Tony | 7/8/2015 | | Gel of Gibson Results LiP, MnP, AKR, VP | Erich, Suraj, Melissa | 7/9/2015 | | LB broth with amp | Bo, Lexi, Kate | 7/9/2015 | | Ligation of promoter+rbs and lysing agent+terminator | Bo, Lexi, Kate | 7/9/2015 | | E. Coli Transformation of Promoter-RBS and LysingAgent-Terminator Ligations | Bo, Lexi, Kate | 7/10/2015 | | Gibson Assembly of PCR purified VP1 + VP2 | Jill, Erich, Melissa, Suraj | 7/10/2015 | | Gel of Gibson Results VP | Jill, Erich, Melissa, Suraj | 7/10/2015 | | Ethanol Precipitation of VP1 and VP2 | Jill, Erich, Melissa, Suraj, Mark | 7/10/2015 | | Non-inoculated yeast plates test | Tony | 7/10/2015 | | Inoculation of Promoter+RBS and LysingAgent+Terminator E. Coli | Bo, Lexi, Kate | 7/12/2015 | | Preparation of Yeast glycerol stocks for long-term storage | Mark, Erich | 7/13/2015 | | Miniprep of promoter+rbs and lysing agent+terminator | Bo, Lexi, Kate | 7/13/2015 | | Gibson Assembly of Ethanol Precipitated VP1+VP2 | Jill, Erich, Melissa, Suraj | 7/13/2015 | | Gel of Gibson Results VP | Jill, Erich, Melissa, Suraj | 7/13/2015 | | Inoculation of liquid yeast cultures | Mark | 7/13/2015 | | Second inoculation of liquid yeast cultures for competent cell preparation | Mark | 7/14/2015 | | Transformation of second diluted yeast cultures with pRS313 | Mark, Jill, Erich, Melissa, Suraj | 7/14/2015 | | Inoculation of liquid yeast cultures | Mark | 7/14/2015 | | Dilution (10x, 25x, 50x, 100x) of inoculated yeast cultures | Mark | 7/15/2015 | | Miniprep of promoter+rbs and lysing agent+terminator | Bo, Lexi, Kate | 7/15/2015 | | Digestion of promoter+rbs and lysing agent+terminator | Bo, Lexi, Kate | 7/15/2015 | | Preparation of E. Coli glycerol stocks for P+R and L+T | Bo, Lexi, Kate | 7/15/2015 | | Overlap PCR of MnP1 and 2 (w/ primers and w/o) | Mark, Jill, Erich, Melissa, Suraj | 7/15/2015 | | Gel of PCR Overlap Results | Jill, Erich, Melissa, Suraj | 7/15/2015 | | Ligation of promoter+rbs+lysing agent+terminator | Bo, Lexi, Kate | 7/15/2015 | | Transformation of promoter+rbs+lysing agent+terminator | Bo, Kate, Lexi | 7/16/2015 | | Gel Electrophoresis of MnP Overlap PCR | Mark, Suraj | 7/16/2015 | | Overlap PCR of LiP (straight gBlocks and PCR amplified gBlocks) | Mark, Suraj | 7/16/2015 | | Gel Extraction of MnP | Erich | 7/16/2015 | | Overlap PCR of LiP (straight gBlocks) | Suraj, Tony, Arren, Jill, James | 7/16/2015 | | Gel of Overlap PCR of LiP | Suraj, Jill, Melissa, Erich | 7/17/2015 | | PCR Amplification of Lac1, Lac2, Tyr2 | Suraj, Jill, Melissa | 7/20/2015 | | Overlap PCR Part 2 of MnP | Erich | 7/20/2015 | | Overlap PCR (with calculated Tm) of VP | Suraj, Jill, Melissa | 7/20/2015 | | Ligation of promoter+rbs+lysing agent+terminator | Bo, Lexi, Kate | 7/20/2015 | | Overlap PCR (with calculated Tm) of AKR | Suraj, Melissa, Erich | 7/21/2015 | | Restriction Enzyme Digest and Ligation of MnP to pRS315 | Mark, Erich | 7/21/2015 | | LB w/ chlor made | Bo, Kate, Lexi | 7/21/2015 | | Inoculation of full device and E. coli | Bo, Kate, Lexi | 7/21/2015 | PCR Amplification of LiP1, LiP2, Lac1, Lac2, VP1, VP2, AKR1, AKR2, Tyr1, Tyr2 | Melissa, Jill | 7/21/2015 | | E. coli transformation of MnP + pRS315 | Mark | 7/22/2015 | | Gel of PCR Products (LiP1, LiP2, Lac1, Lac2, VP1, VP2, AKR1, AKR2, Tyr1, Tyr2) | Suraj, Erich | 7/22/2015 | | Gibson assembly of AKR1 + AKR 2 | Jill | 7/22/2015 | | Gel of Gibson Results AKR | Suraj | 7/22/2015 | | PCR Purification of AKR2, LiP2, VP1, VP2 | Jill | 7/22/2015 | | Mini prep + nanodrop | Bo, Lexi, Kate | 7/23/2015 | | Gibson Assembly of VP1 + VP2 | Mark | 7/23/2015 | | Gel Extraction of LiP1, Lac1, Lac2, AKR1, | Suraj, Melissa, Jill | 7/23/2015 | | Gel Tyr1, Tyr2, Lac2, Gibson Assembley of AKR | Suraj, Tony | 7/23/2015 | | Streaking of new Yeast (BY4741) plate | Mark | 7/23/2015 | | EcoRI and PstI digest of AKR and MnP | Mark | 7/27/2015 | Liquid Inoculation of Yeast MST WT Cultures | Erich | 7/27/2015 || Digestion of GFP and full device for insertion into chlor and amp backbone | Lexi, Kate | 7/27/2015 | | PCR of Lac2 and Gibson AKR | Tony, Melissa | 7/27/2015 | | Gel electrophoresis of Lac2 (for extraction), AKR(test), & Tyr1(for extraction) | Tony, Melissa | 7/27/2015 | | Vector Insertion of AKR and MnP into pSCB13C | Mark | 7/27/2015 | | Transformation of AKR and MnP and GFP | Bo, Kate, Lexi | 7/28/2015 | | Yeast Transformation of backbones pRS313, pRS315, and pRS316 | Erich | 7/28/2015 | | LB + Chlor plates made | Tony | 7/28/2015 | | Gel of Lac1 and Lac2 PCR amplified products (140uL in big wells) | Suraj, Melissa | 7/29/2015 | | PCR Purification of LiP Gibson Results | Jill, Melissa | 7/29/2015 | | Digestion of Full Device and yeast backbone | Bo, Lexi, Kate | 8/3/2015 | | Digest of Mnp, AKR, GFP, full device and backbone | Bo, Kate, Lexi | 8/3/2015 | EtBr Gel of (Lane1: Ladder, Lane2: Tyr Gibson 20uL, Lane3: PCRLac1 A 10uL, Lane4: Lac1B, Lane5: Lac1C, Lane6: Lac2A, Lane7: Lac2B, Lane8: Lac2C) | Jill, Erich, Suraj, Melissa | 8/3/2015 || Transformation of AKR, MnP, GFP, and full device | Bo, Kate, Lexi | 8/4/2015 | | Gel of Lac1 and Lac2 PCR amplified products (140uL in big wells) (Lane 1: Ladder, Lane 2: Lac1, Lane3: Lac2) | Jill, Erich, Suraj, Melissa | 8/4/2015 | | Gel of (Lane1: Ladder, Lane2: 5ul Tyr 1 Gel extract, Lane3: 5uL Tyr2 Gel Extract) | Jill, Erich, Suraj, Melissa | 8/4/2015 | | Gel Extraction of PCR amplified Lac1 and Lac2 | Jill, Suraj, Erich | 8/4/2015 | | Gibson Assembly of Tyr1+Tyr2 (different gel extraction than before) | Jill, Melissa | 8/4/2015 | | Gibson Assembly of Lac1 + Lac2 gel extracts | Jill, Melissa | 8/5/2015 | | Gel of Gibson Assembly results (Lane1: Ladder, Lane2: Tyr, Lane3: Lac) | Jill, Suraj, Melissa | 8/5/2015 | | Gibson Assembly of Tyr1+2 and Lac1+2 (with more DNA added) | Jill, Suraj, Melissa | 8/5/2015 | | Gel Extract of Gibson Assembly Results Tyr | Erich | 8/5/2015 | | Re-Gibson Assembly of Lac1 & Lac2 | Jill, Suraj | 8/6/2015 | | PCR of Tyr | Jill, Suraj | 8/6/2015 | Gel of (Lane1: Ladder, Lane 2: Lac Gibson, Lane 3: Lac Gibson, Lane 4: Tyr1, Lane5: Tyr2, Lane6: Tyr3, empty, empty) | Jill, Suraj | 8/6/2015 || Gel of the rest of Tyr (140uL) (Lane 1: ladder, Lane2: Tyr, empty) | Jill, Suraj | 8/6/2015 | | Gel Extraction of Lac Gibson (3.5ng/uL) | Jill, Suraj | 8/7/2015 | | Re-measurement of Lac and Tyr concentrations using Cubit instead of nanometer | Jill, Suraj, Melissa | 8/26/2015 | | PCR of Gibson Tyr and Lac | Jill, Suraj | 8/27/2015 | | PCR of Tyr1, Tyr2, Lac1, Lac2 | Jill, Suraj | 8/27/2015 | | Gel 1: Lane 1: Ladder, Lane 2: Lac1 PCR, Lane 3: Lac2 PCR, Lane 4 Tyr1 PCR, Lane 5 Tyr2 PCR -----Empty | Melissa, Jill | 8/28/2015 | | Gel 2: Lane 1: Ladder, Lane 2: Lac Gibson A, Lane 3: Lac Gibson B, Lane 4 Tyr Gibson A, Lane 5 Tyr Gibson B ---- Empty | Melissa, Jill | 8/28/2015 |