Team:UCLA/Protocols

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Protocols

The following protocols were extensively used over the course of the last iGEM season. Many of these protocols have come from commercial products, and as such will be properly linked.

General Protocols

• Polymerase Chain Reaction (PCR) with Q5 High Fidelity 2X Mastermix (New England Bioloabs): Protocol
• Polymerase Chain Reaction (PCR) with KAPA HiFi Hotstart PCR Kit (Kapa Biosystems): Protocol
• Restriction Enzyme Double Digest (New England Biolabs): Protocol
• Ligation with T4 Ligase (New England Biolabs):Protocol
• Gibson Assembly with GIbson Assembly HiFi 1-Step Kit (Synthetic Genomics, Inc.: Protocol
• Transformation (Electroporation) of E. coli (K12)-C2989K (New England Biolabs) : Protocol
• Making Chemically Competent E. coli (BL21-DE3) (Zymo Research): Protocol
• Transformation (Chemical) of E. coli (B21-DE3) (Zymo Research): Protocol
• Colony Polymerase Chain Reaction (CPCR) with APEX Taq (Apex BioResearch Products): Protocol

Protein Cages Protocols

Protein Expression and Purification

Growth and Pelleting:

1. Inoculate 5 ml of cells in 37 C overnight
2. Incubate cells in larger flask(s) (1 - 4 L per batch) in 37 C until OD reaches 0.8
3. Add 0.5 mM IPTG (1 mL of 0.5 M stock per liter of cells)
4. Incubate cells in 30 C for 5 hrs
5. Aliquot cells into 50 mL Falcon tubes
6. Wash and pellet cells
   1. Centrifuge cells 4000g for 10 min
   2. Dump out supernatant
   3. Resuspend cells in 10 ml of cold water
   4. Consolidate suspended pellets into fewer falcon tubes
   5. Centrifuge… Repeat until all cells washed into a single tube

Lysis and Filtration:

1. Resuspend cell pellet in lysis buffer (1 : 2 cell weight : ml of lysis buffer)
   1. PCQuad: 20mM sodium phosphate (ph=8.0), 300 mM sodium chloride, 10 mM imidazole
   2. O3-33: 50 mM TRIS (ph=8.0), 250 mM NaCl, 20mM imidazole
2. Chill cell suspension for 10 min
3. Add 10 uL 100 mM PMSF for every mL of cell suspension right before sonication
4. Sonicate cell suspension:for 10 sec (10 cycles; 30 sec pause between each cycle)
5. Centrifuge lysates for 30 min at 13000 rpm
6. Filter supernatant through a 22 um Millipore filter

HisTag Purification and SDS-PAGE:

1. Used Hispur column purification protocol
   1. Add appropriate amount of resin bed to purification column
   2. Equilibriate column with two resin bed volumes of equilibration buffer
   3. Add equal volume of equilibration buffer through filtered protein sample
   4. Add sample through column. Collect flow through and reapply through column. Save flow through for downstream analysis if desired
   5. Wash resin with two resin bed volumes of wash buffer with a gradient of imidazole concentrations until protein A280 reaches baseline Save flow through for downstream analysis if desired
   6. Elute protein with two resin bed volumes of elution buffer. Repeat elution. Save flow through for downstream analysis if desired
   7. Imidazole concentrations used:
      1. Equilibration: 20 mM
      2. W1: 25 mM, W2:40 mM, W3: 150 mM
      3. E1: 400 mM, E2: 400 mM
2. Run SDS-PAGE on equilibration, three washes and two elutions
   1. Mix samples with an equal volume of sample buffer (prepped with 2-B-mercaptoethanol)
   2. Heat samples at 80 C for 10 min before loading into gel
   3. Use 1x SDS MOPS Running Buffer
3. Run Native-PAGE gel on best elutions
   1. Use MOPS Running buffer
4. Syringe filter 1 mL of best elutions through 0.22 um filter and use 30 uL for DLS

Thrombin Cleavage Assay

1. 60 uL 0.2 mg/ml protein cage samples used for thrombin assay
   
2. For each protein cage tested, a sample was subject to thrombin while another was not
   
3. Added 1.25 uL of 460 units/ml bovine thrombin stock to each sample (0.69 units) to each sample that was to be subject to thrombin every 4 for hours for 16 hours
   
4. An SDS-PAGE gel was run to assay for cleavage and a DLS test was conducted to assay for protein cage disassembly
   
   

Super Soldier Systems (CDI) Protocols

Competition Assay Protocol

1. Grow all cells to correct growth phase in appropriate antibiotic
   1. Positive control to OD600 = 0.35 in 50 mL Luria-Bertani broth (LB) with antibiotic at 37 C
   2. Inhibitor to OD600 = 0.35 in 50 mL Luria-Bertani broth with antibiotic at 37 C
   3. Negative control to OD600 = 0.35 in 50 mL Luria-Bertani broth at 37 C
   4. Target overnight (16 hours) in 50 mL Luria-Bertani broth with antibiotic at 37 C (Target OD600 is not important)
2. Mix inhibitor (or control) and target cell at a ratio of 10:1 (determined through concentration relation listed below) in LB without any antibiotic
   1. Multiply the Mass Attenuation Constant for the bacteria with the reading obtained from the OD600 to get the cellular density of the target, use this to prepare a 10:1 ratio of inhibitor to target cells.
3. Incubate at 37 C at 225 rpm while plating samples at 0 and 4 hours after mixing
4. Plate ten-fold serial dilutions from 1:1 up to 1,000,000 (experimentally determine which dilutions are best for your needs) in a M9 salt solution or SOC
1. Plate 100 µL
5. Count Colony Forming Units per mL of the co-culture
   1. Choose plates that haven’t formed lawns, you want easy to identify colonies ideally
   2. Count the number of colonies on that plate (choose a dilution in which all three co-cultures can be counted)
   3. Divide the number of colonies by the dilution factor to get the number of CFUs
   4. Divide that value by the fraction of mLs plated to get CFUs/mL
6. Compare the CFUs of the inhibitor with the positive and negative control to determine if the inhibitor properly inhibits the target strain