iGEM 2017 — Team iTesla-SoundBio

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# Team:iTesla-SoundBio/Notebook

# Notebook

**Date:** 5/28/2017

* Amplified pcbA1 with normal primers
* MEA, JA

**Date:** 6/04/2017

* Ran gel for future purification on Gibson PCRs conducted last training session, and isolated pcbA1 DNA
* KZ, MEA, JJW

**Date:** 6/23/2017

* Amplified genes pcbA4 and pcbA5 using PCR
* RAN, JA

**Date:** 6/26/2017

* PcbA5 & pcbA4 PCR product Gel Analyzation & Purification, Restriction Digest of pET-29B
* RAN, JA, MEA, JJW

**Date:** 6/30/2017

* Ran analyzation and purification gels on digested pET-29B plasmid
* RAN, JA, MEA, JJW

**Date:** 7/01/2017

* Conducted gel extraction on the pET-29B plasmid and pcbA4 gblock that were caught in previous gel electrophoresis slices
* JJW, MEA

**Date:** 7/08/2017

* Used a nanodrop to measure purity and concentration of DNA in digested pET-29B and pcbA4 samples that underwent gel purification
* KZ

**Date:** 7/10/2017

* pcbA4 pET-29B Gibson Assembly
  MEA, JA, JJW, KZ

**Date:** 7/13/2017
Transforming Plasmids into Purchased Competent Cells

* MEA, JJW, KZ

**Date:** 7/16/2017

* Inoculating Transformed Purchased Cells into LB+kan Broth
* ZM, MEA
* Transforming pcbA4 Plasmids into Self-Made Competent Cells
* KZ, MEA

**Date:** 7/18/2017

* Inoculating Transformed Self-Made Cells and pET-29B Plasmids
* JA, MEA, KZ

**Date:** 7/23/2017

* Miniprep of Inoculated Cells and Nanodrop Verification
* MEA, JA, KZ

**Date:** 7/25/2017

* Digesting pET-29B Plasmids
* KZ

**Date:** 7/30/2017

* Gel Purifying Digested pET-29B and Inoculating Transformed Cells
* MEA, KZ

**Date:** 7/31/2017

* Gel Extraction of Digested pET-29B and Nanodrop Verification
* MEA, KZ

**Date:** 8/1/2017

* Gibson Assembly pcbA4 gene (insert) into digested pET-29B plasmid (vector)
* KZ

**Date:** 8/5/2017

* Transformation of Gibson Assemblied pcbA4 Plasmids into Self Made Competent Cells
* MEA

**Date:** 8/8/2017

* Creating Carb plates
* JJW

**Date:** 8/9/2017

* PCR to amplify pcbA4 geneblock
* JJW

**Date:** 8/11/2017

* Colony PCR on transformed e.coli cells containing pcbA4
* KZ

**Date:** 8/12/2017

* Gel analysis on our plasmids that have underwent colony PCR
* KZ, RAN

**Date:** 8/14/2017

* Gel electrophoresis on PCRed pcbA4 gblock and colony PCR trial #7 and #8
* JJW

**Date:** 8/19/2017

* Prepping DH5 alpha Cells for Transformation
* JJW, MEA

**Date:** 8/20/2017

* Gibson Assembly and Transformation of Positive Control
* KZ

**Date:** 8/30/2017

* Amplifying Geneblock with PCR and Gel Analyzation of PCR
* MEA, KZ

**Date:** 8/31/2017

* Digesting pET-29B Plasmids and Gel Analysis
* MEA, KZ

**Date:** 9/10/2017

* Amplify all three PCB geneblocks through PCR and purify the PCRed solutions through PCR purification
* MEA, SA, JJW, KZ

**Date:** 9/12/2017

* Pet-29b and Biobrick Backbone Miniprep, Analytical Gel on PCR geneblocks and vectors
* JA, MEA

**Date:** 9/15/2017

* Restriction Digest on PCRed A1, 4, 5 and Miniprepped Vectors/ PCR on A4/5 Promoter and A5 gblock
* MEA, JJW, MS

**Date:** 9/20/2017

* PCR Purify and Gel Analyze pcbA5 and A4/5 Promoter, Gel Purify Digested Tubes
* MEA, JA, AC, JJW, KZ

**Date:** 9/27/2017

* Gel Extracting Biobrick Ingredients and Digesting Promoter and pcbA5
* MEA, JA, CL, JJW, KZ

**Date:** 9/29/2017

* Gel Purify and Gel Extract A4/5 Promoter, pcbA5, and pSB1C3
* JJW, MEA, SA

**Date:** 10/4/2017

* Ligation and Transformation of bb and e Backbones and gblocks
* JJW, JA, MEA

**Date:** 10/8/2017

* Miniprep of Transformed RFP Backbone Colonies
* JJW, KT, MS

**Date:** 10/11/2017

* Restriction Digest on RFP and Running Purification Gel, Preparing Mini Preps for Sequencing
* MEA, CA, AC, JJW, JA

**Date:** 10/13/2017

* Gel Extraction of RFP, PCR Promoter and AmilGFP with primers, Making Antibiotic Plates
* JJW, MEA

**Date:** 10/17/2017

* New PCR on A5 and Promoter Inserts
* JA

**Date:** 10/18/2017

* Ligation and Transformation of Plan A, Digest and Gel Purification of Plan B, Off-the-cuff Digest of Promoter and RFP
* JJW, JA, CL, MEA

**Date:** 10/20/2017

* Miniprepping Successfuly Transformed Colonies
* JJW, MEA, JA, SA

**Date:** 10/21/2017

* Preparing Miniprep Samples for Sequencing
* JA, JJW

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# Team:iTesla-SoundBio/Experiments

# Experiments

### PCR (50 uL reaction):

1. 0.5 uL of DNA
2. 1 uL of 10 uM forward primer
3. 1 uL of 10 uM reverse primer
4. 35.5 uL of ddH2O or RNase-free water
5. 10 uL Phusion 5X Buffer. Keep on ice.
6. 1 uL Phusion DNA polymerase. Keep on ice.
7. 1 uL dNTPs
8. Quick spin to make sure all the liquid is at the bottom

#### Program:

Lid Temp: 100 C

1. 98 C for 30 sec
2. 30 Cycles:
   1. 98 C for 10 sec
   2. 60 C for 20 sec
   3. 72 C for 60 sec
3. 72 C for 120 sec

### Gel Electrophoresis:

1. Make 1% TAE and agarose solution:
   1. For 100 mL, add 1g of agarose to TAE
   2. Microwave until agarose is fully dissolved and solution is clear
   3. Once solution has cooled sufficiently, add gel stain. If working with 10,000X, for example, add 10 uL of gel stain.
2. Pour into tray and wait for it to solidify
3. Place solid gel in gel box and fill with TAE
4. Add loading dye to sample. If working with 5X dye, for example, add 5 uL of dye to 20 uL of the sample.
5. Run sample at 120-170 V until the dye line is about halfway to two thirds down the gel. Keep the size of the sample in mind when running the gel.

### Gel extraction and Purification:

Using QIAquick Gel Extraction Kit

1. Cut out desired gel slice with clean razor blade.
2. Add 800 uL of Buffer QG to tube with gel slice.
3. Incubate tube in water bath at 55 C until gel is fully dissolved. Vortex every 2-3 mins to help it dissolve.
4. Transfer 650 uL of QG + DNA to spin column inside collection tube. Centrifuge for for 30 seconds at 5,000 rpm. Pour out flow-through and repeat until all DNA has been centrifuged.
5. Add 750 uL of buffer PE to spin column. Centrifuge for 30 seconds at 5,000 rpm again. Pour out flow-through.
6. Centrifuge for another 2 minutes.
7. Transfer to new microcentrifuge tube and add 30 uL of ddH2O to the column. Let sit for five minutes and then centrifuge for one minute at 5,000 rpm. Dispose of the spin column.

### Miniprep:

Using Epoch Life Sciences miniprep kit

1. Before starting:
   1. Add provided RNase A solution to Buffer MX1. Store at 4 C.
   2. Add 100% ethanol to Buffer WS and WN.
2. Pellet prepared cells by centrifuging for 1 min. Remove all supernatant liquid.
3. Completely resuspend pelleted cells in 200 ul of MX1 Buffer.
4. Add 250 uL of MX2 and gently mix (invert the tube 6 times) to lyse the cells. Incubate at room temperature for no more than 5 mins.
5. Add 350 uL of MX3 to neutralize the lysate, and gently mix the solution immediately.
6. Centrifuge for 10 minutes at 13,000 rpm.
7. Transfer the supernatant into a column inside a collection tube.
8. Centrifuge for 30 seconds at 5,000 rpm. Pour out flow-through.
9. Wash by adding 500 uL of WN Buffer and centrifuging for 1 min at 9,000 rpm. Pour out flow-through.
10. Add 700 uL of WS Buffer and centrifuge for 1 min at 9,000 rpm. Pour out flow-through.
11. Centrifuge for another 2 mins at 13,000 rpm to remove residual ethanol.
12. Transfer spin column to a new microcentrifuge tube. Add 50 uL of elution buffer and let sit for 5 mins. Centrifuge for 1 min at 13,000 rpm and dispose of spin column.

### Restriction Digest (40 uL reaction):

1. Biobricking:
   1. Insert:
      1. 34 uL of PCR product (or however much is available, use ddH2O to make up volume needed)
      2. 4 uL of NEB 2.1 buffer
      3. 1 uL XbaI
      4. 1 uL PstI
   2. Vector:
      1. 14 uL of miniprep
      2. 20 uL ddH2O
      3. 4 uL of NEB 2.1 buffer
      4. 1 uL XbaI
      5. 1 uL PstI
2. Pet-29b Backbone:
   1. Insert:
      1. 34 uL of PCR product
      2. 4 uL of NEB 2.1 buffer
      3. 1 uL NdeI
      4. 1 uL XhoI
3. Vector:
   1. 14 uL of miniprep
   2. 20 uL ddH2O
   3. 4 uL NEB 2.1 buffer
   4. 1 uL NdeI
   5. 1 uL XhoI

### Ligation (10 uL reaction):

1. 5 uL of cut and purified PCR product
2. 1 uL of cut and purified vector
3. 2 uL of ddH2O
4. 1 uL of 10X Ligase buffer. Keep on ice.
5. 1 uL of Ligase enzyme. Keep on ice.
6. Incubate at room temperature for 10 mins before transformation.

### Transformation:

1. Prepping overnight chemically competent NEB5 alpha cell cultures:
   1. Add 2 mL of LB to a test tube
   2. Touch p1000 pipette to a colony from agar plate and swirl it in the LB
   3. Leave overnight in shaking incubator at 37 C.
2. After overnight culture is ready, back dilute culture 1:100 so the cells enter log phase. It will take about 2-3 hours for the culture to reach mid-log phase.
3. Pellet cells by centrifuging at 4,000 rpm for 1 min.
4. Remove the supernatant and resuspend in 1 mL of chilled 0.1M CaCl2. Repeat the spin.
5. Repeat this step up to 3 times. The final time, resuspend the pellet in only 50 uL of CaCl2.
6. Add DNA to cells and incubate on ice for 30 minutes.
7. Heat shock by placing incubating tubes in water for 45 sec at 42 C.
8. Immediately return tubes to ice for another 2 mins.
9. Add 500 uL to cells and put in shaking incubator for 1 hour at 37 C.
10. Plate transformed cells on agar plates with appropriate antibiotics. Use ~10 sterile glass beads to spread the liquid around the plate.
11. Incubate plates upside down overnight in shaking incubator at 37 C.

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# Team:iTesla-SoundBio/Results

# Results

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