Team:Washington/Notebook

A DETAILED DESCRIPTION OF THE PROTCOLS WE USED

• Gene Synthesis (DONE)
• Colony PCR (DONE)
• Assembly
• Cloning (??)
• Expression (DONE)
• Purification (DONE)

Protein Gel Microscope

Flow Cytometry

1. Set up overnights of parts 48-51. Let grow overnight.
2. Dilute 1 ul overnight into 1ml
3. Add 1 mm IPTG and let grow for four hours
4. After cells have grown up, place in flourophore (1 um) and allow time to bind (1 hour)
5. Also place 1 ul beads in 1 ml along with 1 ul flourophore.
6. Allow beads to bind to flourophore, then spin beads down, remove supernatent and replace with 1 ml water.
7. Read the samples through the flow cytometer

Supernatant Protein Purification, 50mL

1. Inoculate 50mL culture of TB with ~750uL overnight culture
2. Grow a 37c until OD600: 0.4
3. Inoculate cells with IPTG so that the final concentration is 0.5mM (25uL of 1M IPTG for 50mL culture)
4. Grow cultures until OD600: 4, or use time points if looking for comparison in protein in supernatant
5. Transfer culture to 50mL Falcon Tube
6. Centrifuge at 8000rpm for 20 min to pellet cells
7. Pour supernatant into 60mL syringe with 0.45uM filter attached
8. Apply filtered supernatant to Ni-column (see Ni-column Set up for directions)
9. Collect flow through and re-apply to column
10. Wash column with 12mL PBS
11. Elute column into 15mL condenser tube with 12mL PBS with 100mM imidazole
12. Spin elutant in condenser tube until desired volume (~500uL) this is you purified protein.

Ni-column Set up

1. Transfer 2mL NiNTA beads (Quigen) to column
2. wash column with 10mL dH2O
3. Equillibrate column with 10mL running buffer (usually PBS)
4. To reuse column
   1. Wash with 12mL dH2O
   2. Wash with 12mL 100mM EDTA
   3. Wash with 12mL dH2O
   4. wash with 12mL 100mM Ni(SO4)
   5. Wash with 12mL dH2O
   6. Add 12mL 20% Ethanol run till ~5mL remains in column
   7. cap for use later

Supernatant Protein Purification, 2mL

1. Inoculate 50mL culture of TB with ~750uL overnight culture
2. Grow a 37c until OD600: 0.4
3. Inoculate cells with IPTG so that the final concentration is 0.5mM (25uL of 1M IPTG for 50mL culture)
4. Grow cultures until OD600: 4, or use time points if looking for comparison in protein in supernatant
5. Prep NiNTA columns
   1. Place micro-centrifuge columns in collection tubes
   2. In micro-centrifuge columns add 200uL NiNTA beads
   3. Add 500uL PBS, aspirate to thoroughly rinse columns
   4. Spin columns with collection tubes for 30sec at 500rpm
6. Transfer 2mL of growing culture to eppendorf tube
   1. Spin tube for 20min at 8000 rpm
   2. Remove supernatant, carefully as to not disrupt the pellet and set aside
7. Bind Protein to column
   1. Add 500uL supernatant to the column
   2. Spin for 30sec at 500rpm
   3. Discard flow through
   4. Repeat 1-3 until all supernatant has run though the column
8. Wash column
   1. Apply 500uL PBS to column, aspirate to suspend beads
   2. Spin column for 30sec at 500rpm, discard flow through
   3. Repeat 1-2
9. Elute protein off of column
   1. Make PBS with 1mg/mL BSA and 100uM imidazole
   2. Add 200uL PBS + 1mg/mL BSA + 100uM imidazole to column aspirate to mix beads
   3. Let sit for 2 min
10. Place Column in clean collection tube
11. Spin column for 5min at 500rpm
12. FLOW THROUGH IS YOUR PURIFIED PROTEIN

Gene Synthesis

1. Generate Oligo's
   1. Go to: http://helixweb.nih.gov/dnaworks/
   2. Set parameters
      1. Enter you job title and email
      2. Choose E.Coli Class II for codon frequency
      3. Set Annealing temperature to 60
      4. Maximize oligo length for cheapest oligo (60 for most companies)
      5. Set Number of solutions = 10
      6. Select Non-degenerate sites to avoid
         1. Bio-Brick requires EcoRI, XbaI, SpeI, and PstI, others can be chosen if desired
      7. Leave rest of options default
   3. Enter Sequences
      1. Click "Add Sequence Field" twice under Sequence formats
      2. Imput your header sequence (select Nucleotide, this contains your cut sites,spacers, etc for subsequent cloning)
         1. eg. GGATAGGA CATATG
      3. Enter you protein sequence (select protein)
      4. Imput your tail sequence (select Nucleotide, this contains your cut sites,spacers, etc for subsequent cloning)
         1. e.g. CTCGAG ATTCGATG
   4. RUN
      1. If nothing is running make sure there are no blank new lines in your sequence section!
   5. Choose your favorite oligo set to synthesize your gene
      1. Usually look for the best scoring with the closest Tm's and oligo lengths
   6. Design two additional oligos to amplify your gene
      1. A FORWARD and REVERSE oligo that complements your final DNA sequence with a Tm of 65. Just copy from the 5’ end of your first and last oligo from oligo’s reported from DNAWorks until you have a calculated Tm of 65 (20‐30bp, +/‐ 1deg). Try to make sure then ends are either G/C.
   7. ORDER
      1. I often try and order in plates (easier if ordering a lot) and make sure that the nmol of oligo is normalized. For IDT this is free, but that may differ for other companies.
2. Synthesize Gene
   1. Dilute all oligos to 100uM
   2. Mix together
      1. add 5uL of each into a new master tube
   3. Setup Synthesis PCR Reaction (have tried Taq, Vent, and PfuTurbo. Results are always best with Phusion)
      1. 1uL Oligo Mix
      2. 1uL 25mM dNTP's
      3. 10uL Phusion HF Buffer
      4. 0.5uL Forward Oligo
      5. 0.5uL Reverse Oligo
      6. 0.5uL Phusion polymerase
      7. 36.5uL diH2O
   4. Synthesis PCR Reaction
      1. 98C - 30s
      2. 98C - 10s
      3. 63C - 10s
      4. 72C - 30s/kb target gene
      5. Repeat 2-4 29x
      6. 72C - 5min
      7. 10C - forever
   5. Setup Amplification PCR Reaction
      1. 1uL FROM UNPURIFIED SYNTHESIS REACTION
      2. 1uL 25mM dNTP's
      3. 10uL Phusion HF Buffer
      4. 0.5uL Forward Primer (Tm 65)
      5. 0.5uL Reverse Primer (Tm 65)
      6. 0.5uL Phusion polymerase
      7. 36.5uL diH2O
   6. Amplification PCR Reaction
      1. 98C - 30s
      2. 98C - 10s
      3. 63C - 10s
      4. 72C - 30s/kb target gene
      5. Repeat 2-4 29x
      6. 72C - 5min
      7. 10C - forever
3. Run a 1% agarose gel of the synthesis and amplification reaction
   1. 5uL sample, 1uL loading buffer
   2. You should see a smear from 60bp to over your gene length in the synthesis reaction
   3. In the Amplification reaction a single band with your gene of interest should be there
4. Continue on with standard cloning!
   1. Make sure to sequence at least 4 clones. Often all 4 will be correct, but insertions,deletions, and spurious mutations sometime occur during the synthesis protocol.
5. TROUBLESHOOTING
   1. Often I focus on the amplification step, assuming that there is a smear for the synthesis step on the gel and that smear covers the size of your gene of interest.
   2. First I often remove the annealing step use a 2 step protocol (Denature – Amplify x 29)!
   3. Still, if no gene is amplified I run a gradient PCR
   4. Then I try 0.5M Betaine (from 5M stock), or 5% DMSO
   5. Finally if nothing is working I break the gene into chunks and amplify smaller sections, then add those sections together and try to amplify the entire gene from the larger chunks.

Colony PCR

1. Prepare one sterile 0.6mL tube with the following reaction mixture for each colony you intend to pick.
   1. 5uL Qiagen Master Mix
   2. 1uL 40uM VF2
   3. 1uL 40uM VR
2. Prepare one sterile 0.6mL tube with 20uL sterile diH2O for each colony you intend to pick.
3. Pick colonies
   1. Pick a single colony using a micropipettor with sterile tip. The pippettor should be set to 3uL
   2. Aspirate colony into 20uL diH2O vigorously to transfer cells to diH2O
   3. Transfer 3uL of diH2O containing cells to reaction mixture set up in step 1
4. Run reaction
   1. 94C - 3min
   2. 94C - 30s
   3. 55C - 30s
   4. 72C - 1min / kb gene
   5. repeat 2 - 4 29x
   6. 72C - 10min
   7. 4C - forever

Gene Assembly using the NheI and PstI sites

1. Start with first 23 coding nucleotides of gene, eg. 5'-atgcgtaaaggagaagaacttt...-3'
2. Replace the atg start codon with the XbaI site: 5'-TCTAGA-3', eg. 5'-TCTAGAcgtaaaggagaagaacttt-3'
3. Add 6-8 random nucleotides to the 5' end of the primer, eg. 5'-cgggcTCTAGAcgtaaaggagaagaacttt-3'
4. Tweak the 3' end of the primer (add /remove nucleotides) so that the annealing temperature is close to that of VR
5. Amplify your gene using the designed forward oligo and VR
6. PCR purify the PCR product
   1. To ensure that the proper size fragment was amplified 5uL of PCR reaction can be run on an agarose gel
7. Digest PCR product with XbaI and PstI
   1. PCR purify
8. Digest Vector with NheI and PstI
   1. PCR purify
9. Mix insert and vector in 3:1 ratio and ligate
10. Transform into competent cells
11. Screen cells for correct insert using VF2 and VR

Gene Assembly using the NheI sites

1. Start with first 23 coding nucleotides of gene, eg. 5'-atgcgtaaaggagaagaacttt...-3'
2. Replace the atg start codon with the XbaI site: 5'-TCTAGA-3', eg. 5'-TCTAGAcgtaaaggagaagaacttt-3'
3. Add 6-8 random nucleotides to the 5' end of the primer, eg. 5'-cgggcTCTAGAcgtaaaggagaagaacttt-3'
4. Tweak the 3' end of the primer (add /remove nucleotides) so that the annealing temperature is close to 58C
5. Next start with the last 23 coding nucleotides eg.5'-tattttcagggtgctagctaa-3'
6. Remove the stop codon(s), in this case taa, and replace with the SpeI cut site ACTAGT, eg. 5'-tattttcagggtgctagcACTAGT-3'
7. Add 6-8 random nucleotides to the 3' end of the primer, eg. 5'-tattttcagggtgctagcACTAGTctgggtc-3'
8. REVERSE COMPLEMENT PRIMER, eg. 5'-tattttcagggtgctagcACTAGTctgggtc-3' --> 5'-gacccagACTAGTgctagcaccctgaaaata-3'
   1. Tweak the 3' end of the primer (add /remove nucleotides) so that the annealing temperature is close to 58C
9. Amplify your gene using the designed forward and reverse primers
10. PCR purify the PCR product
    1. To ensure that the proper size fragment was amplified 5uL of PCR reaction can be run on an agarose gel
11. Digest PCR product with XbaI and SpeI
    1. PCR Purify
12. Digest Vector with NheI and CIP
    1. PCR purify
13. Mix insert and vector in 3:1 ratio and ligate
14. Transform into competent cells
15. SCREEN CELLS FOR CORRECT INSERT ORIENTATION by colony PCR using VF2 and custom reverse oligo

A ROUGH TIMELINE OF THE PROJECT!