Team:Washington/Notebook

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Gram Positive

07/06/10

  • Kunkel Mutagenesis

07/07/10

  • Stained and scanned protein gel. See Chris' notes on [Friday 7/2/10][http://soslab.ee.washington.edu/igem/2010/index.php/Chris#Friday_7.2F2.2F10] for contents and order.

BioRad Micro Column Protein Prep: Expression

  • Arctic Express 13C 24H no anti, Arctic Express 13C 24H kan, DE3* 22C 24H kan, Arctic Express 37C 24H Kan. (Both CapD and CapDCP)
    • CapD 22C kan and 37C kan grew much slower than the rest. (Takes about 3 - 4hrs vs around 1.5-2 hrs for the rest.)
      • One of the two actually decreased OD after 30 minutes.
  • Induced with IPTG after reached OD600 between .7-1.0 (CapD 22C kan and 37C reached about 1.3) and incubated at given temperatures.

07/08/10

  • Made glycerol stocks for Kunkel's. Put in -80C Freezer. To be shipped for sequencing.
  • Spin down remaining proteins(CapD Artic no Anti, CapD CP Arctic no Anti, CapD* Kan 22, CapD CP* Kan 22, CapD Kan Artic, CapD CP Kan Arctic) and place in -20C, awaiting purification.

07/09/10

Protein Purification

  • Lysis
  • Bind Protein
    • Numbers 6 and 7, CapD CP Anti and CapD CP 22, respectively, had lysate form in the supernatant that had been filtered through the TALON beads.
    • Sometimes centrifuged twice after pouring out supernatant because a significant amount had not filtered through TALON beads
  • Wash Protein
    • Sometimes centrifuged twice after pouring out supernatant because a significant amount had not filtered through TALON beads
  • Elution

(Note: Desired proteins have excess histidine chain that binds to TALON beads. Imidazole has higher affinity for TALON beads, so higher imidazole concentration pushes desired protein off, allowing it to fall through filter. This is why higher and higher concentrations of imidazole are used. Cleans off undesired proteins first all the way up to, hopefully, only the desired protein.)

Electrophoresis

  • Ran two gels. #1 has ladder in well 1. #2 has ladder in well 2. 3 wells per condition being Lysate, Supernatant, Purified, in that order. SDS 6uL+6uL of substance for mix. 10uL per well.

Gel #1

  • CapD Arctic No Anti, CapD Arctic Kan, CapD* Kan 22, CapD CP* Kan 37

Gel #2

  • CapD CP Arctic No Anti, CapD CP Arctic Kan, CapD CP* Kan 22, CapD* Kan 37

07/12/10

Nano Drop (Bradford Protein Assay)

  • Create normal line with known concentrations of protein (9uL Coomassie dye, 1uL protein solution) (We used .25mM, .5mM, 1mM, 2mM)
  • Use 1uL of each protein sample with 9uL Coomassie dye and measure with program.

Overnight Glycerol Stocks

  • CapD/CapD CP TB (LB?) Kan solutions with a dab of glycerol from -80C (just scrape glycerol with pipette tip and drop into TB Kan solution).
  • Incubate in 37C shaker overnight.

Dialysis

  • Put proteins into dialysis tubes. Left in cold room overnight. (Used to reduce amount of imidazole in solution.)
    • 1. CapD Arctic no Anti
    • 2. CapD Arctic Kan
    • 3. CapD* Kan 22
    • 4. CapD* Kan 37
    • 5. CapD CP Arctic no Anti
    • 6. CapD CP Arctic Kan
    • 7. CapD CP* Kan 22
    • 8. CapD CP* Kan 37

07/13/10

Nano Drop (Bradford Protein Assay)

  • Run Nano drop to find mg/mL post-dialysis

Enzyme Assay

  • Diluted small amount of each dialyzed enzyme for Assay reaction components
  • Ran fluorescence test with plate reader
  • Got weird results (low, delayed, or no activity)

Post Assay Electrophoresis

  • Ran gel to determine protein concentrations using 1mg/mL, .5mg/mL, .25mg/mL, .125mg/mL, .0625mg/mL, and .03125mg/mL BSA as standard.
  • Will enter order of wells tomorrow once paperwork is on hand

Post-assay Gel order

  • 1) Ladder
  • 2) 1mg/mL BSA
  • 3) 0.5mg/mL BSA
  • 4) 0.125mg/mL BSA
  • 5) 0.25mg/mL BSA
  • 6) 0.06mg/mL BSA
  • 7) 0.3mg/mL BSA
  • 8) CapD Arctic no anti
  • 9) CapD Arctic Kan 13C
  • 10) CapD* Kan 22C
  • 11) CapD* Kan 37C
  • 12) CapD CP Arctic no anti
  • 13) CapD CP Arctic Kan 13C
  • 14) CapD CP* Kan 22C
  • 15) CapD CP* Kan 37C

MiniPrep

  • Done by David and Chris to obtain plasmids for PCR

7/14/10

PCR (Polymerase chain reaction)

  • Diluted Primers to 10uM (10uL of 100uM primer with 90uL of diH2O)
  • Created mixes using the recipe from Table 1a at [http://www.neb.com/nebecomm/products_intl/protocol210.asp]
    • Primers were CapDOS, CapNSS
    • Template DNA was from MiniPrep (Plasmids)
    • Added T7 reverse primer to both
    • Added Phusion DNA Polymerase last to delay reaction

Kunkel Mutagenesis Sequence

  • Checked sequences that came back for desired mutations using CLC Sequence viewer [http://www.clcbio.com/index.php?id=479]. See Google Docs form for mutation information.[http://spreadsheets.google.com/ccc?key=0AvA_ILRbNLPfdEdDUk90ZFBYYjdpU1lCTHNmRl9kMFE&hl=en#gid=0]

Overnights

  • Made 6 overnights from glycerol stocks in -80C with TB Kan CapD CP*, one control, two mutant catalytic knock outs (T2A and T2V), and three mutants (F24Y, L40R, S143R)
  • Incubate at 37C in iGEM lab

Gel

  • Dialyzed proteins seemed to be very low in concentration. (BSA standard might be twice the labeled concentration on gel)


7/15/10

IPTG Induction

  • Pulled overnight glycerol stocks to be put into TB Kan solution
  • Added 1mL of culture per 50mL flask of TB Kan, 10mL of culture per 500mL flask of TB Kan
  • Incubated at 37C until OD600 was 0.6-0.8.
  • Flasks were all incubated at 37C after induction, except the control, for about 1 hour before being moved to 22C.

7/16/10

Scan Gel

  • Post Assay Gel scanned

Colony PCR

  • Boil cells with PCR with "green mix" and +T7/-T7 primers.
  • Run on DNA gel to see if mutation occurred (determine by band size).

Spin down cells

  • 4000rpm for 20 minutes
  • Store at -20C until purification

7/26/10

Induction

  • Grew up T20S, T2V, T59N, T59Q, T59H_M61A, T59H_M61T at 37C to OD0.5-0.8
  • Induced with 500uL IPTG
  • Continue incubation for 24hrs at 22C

7/27/10

  • Concentrated purified proteins
  • Ran gel
  • Ran Bradford and Enzyme Assays
  • Spin down T20S, T2V, T59N, T59Q, T59H_M61A, T59H_M61T

7/28/10

Protein Purification

  • T59Q sat in centrifuge overnight at 4C, spun down in morning again.

7/29/10

Electrophoresis

Gel #1

  • Ladder
  • A CapDCP
  • B T2V
  • C T20S
  • D T59H_M61A
  • E T59H_M61T
  • F T59N
  • G T59Q
  • BSA 2mg/mL
  • BSA 1mg/mL
  • BSA 0.5mg/mL
  • BSA .25mg/mL
  • BSA .125mg/mL
  • BSA .0625mg/mL

Gel #2

  • Ladder
  • BSA 2mg/mL
  • BSA 1mg/mL
  • BSA 0.5mg/mL
  • 1 CapD
  • 2 CapDCP
  • 3 CapDNSS
  • 4 CapDOS
  • 5 F24Y
  • 6 L40R
  • 7 S143R
  • 8 S22I,T59Q
  • 9 S57H,M61H
  • 10 T2A
  • 11 T2V

Notes

  • De-staining started at 4 pm
  • Used up remaining possibly "bad" gels. Gels have large blue blotch at the bottom.

Enzyme Assay

  • Row A=H2O
  • Row B=L-Glutamate
  • 1 CapDCP
  • 2 T2V
  • 3 T20S
  • 4 T59H_M61A
  • 5 T59H_M61T
  • 6 T59N
  • 7 T59Q
  • 8 diH2O

7/30/10

Kunkel Mutagenesis

  • 1. CapDCP NSS
  • 2. CapDCP NSS Foldit
  • 3. CapDCP w/ Thrombin
  • 4. F24H, R356K
  • 5. G79A
  • 6. M61H
  • 7. M61N
  • 8. S22Q, T59Q
  • 9. T18L, T59Q_M61N, F452W
  • 10. T20S, T59Q_M61T
  • 11. T59Q_M61Q, F452W
  • 12. T59S_M61H
  • 13. H2O

8/2/10

  • First Kunkel's failed for unknown reasons

Kunkel Mutagenesis Attempt #2

  • 1. CapDCP NSS
  • 2. CapDCP NSS Foldit
  • 3. CapDCP w/ Thrombin
  • 4. F24H, R356K
  • 5. G79A
  • 6. M61H
  • 7. M61N
  • 8. S22Q, T59Q
  • 9. T18L, T59Q_M61N, F452W
  • 10. T20S, T59Q_M61T
  • 11. T59Q_M61Q, F452W
  • 12. T59S_M61H
  • 13. H2O

8/3/10

  • Picked Kunkel's colonies and placed into plate with LB+Kan culture.
  • Shaking in 37C room
  • Scan Gels from 7/29/10

Well Order (Four per mutation)

  • CapDCP NSS- A1-D1
  • CapDCP NSS Foldit- E1-H1
  • CapDCP w/ Thrombin- A2-D2
  • F24H, R356K- E2-H2
  • G79A- A3-D3
  • M61H- E3-H3
  • M61N- A4-D4
  • S22Q, T59Q- E4-H4
  • T18L, T59Q_M61N, F452W- A5-D5
  • T20S, T59Q_M61T- E5-H5
  • T59Q_M61Q, F452W- A6-D6
  • T59S_M61H- E6-H6

8/4/10

  • Took Kunkel's overnights out of 37C room
  • Sent Glycerol Stocks for Sequencing (100uL 20% Glycerol, 100uL cells)

8/5/10

  • Computational Work from Home

8/6/10

  • Checked all Round 2 sequences. See Google Doc for mutant information.
  • Corrected Data from Assay based on Gel #2 protein concentrations.

8/9/10

  • Autoclave 15 flasks of TB (500mL)
  • Glycerol Stock Overnights (5mL TB+Kan)
    • 1. CapD CP
    • 2. CapD CP w/ Thrombin
    • 3. CapD NSS
    • 4. CapD CP NSS
    • 5. CapD CP NSS Foldit
    • 6. T2V
    • 7. F24H, R356K
    • 8. G79A
    • 9. M61H
    • 10. M61N
    • 11. S22Q, T59Q
    • 12. T18L, T59Q, M61N, F452W
    • 13. T20S, T59Q, M61T
    • 14. T59Q, M61Q, F452W
    • 15. T59S, M61H

Remember to make glycerol stock for T2A and T2V and store in -80C

8/10/10

  • Grow up cells in 500mL TB+Kan
  • Induce with 500uL 1M IPTG once at OD 0.5-0.8
  • Incubate at 18C for 24 hours
    • 1. CapD CP
    • 2. CapD CP w/ Thrombin
    • 3. CapD NSS
    • 4. CapD CP NSS
    • 5. CapD CP NSS Foldit
    • 6. T2V
    • 7. F24H, R356K
    • 8. G79A
    • 9. M61H
    • 10. M61N
    • 11. S22Q, T59Q
    • 12. T18L, T59Q, M61N, F452W
    • 13. T20S, T59Q, M61T
    • 14. T59Q, M61Q, F452W
    • 15. T59S, M61H

G79A had super slow growth rate, about OD 0.1 after 4 hours. T18L, T59Q, M61N, F452W and T59Q, M61Q, F452W about OD 0.3 after 4 hours. G79A completed at 7:00pm.

8/11/10

  • Spin down cells 4000rpm for 20 minutes. Store in -20C.

8/12/10

  • Full Purification Process
  • Concentrate Proteins
  • Run gel of purified/concentrated proteins

8/13/10

  • Interpret protein concentration from gels (scan with Baker Lab scanner)
  • Run enzyme/H2O fluorescence assay
    • CapD NSS ran for 2 possible protein concentrations. One for the bottom two bands, one for all three. Bottom two=3BC, All three=3ABC.

L-Glutamate

      • A1.1 (CapD CP)
      • A2.2 (CapD CP w/ Thrombin)
      • A3.3BC (CapD NSS: lower two bands)
      • A4.3ABC (CapD NSS: All bands)
      • A5.4 (CapD CP NSS)
      • A6.5 (CapD CP NSS Foldit)
      • A7.6 (T2V)
      • A8.7 (F24H,R356K)
      • A9.8 (G79A)
      • A10.9 (M61H)
      • A11.10 (M61N)
      • A12.11 (S22Q,T59Q)
      • C1.12 (T18L,T59Q,M61N,F452W)
      • C2.13 (T20S,T59Q,M61T)
      • C3.14 (T59Q,M61Q,F452W)
      • C4.15 (T59S,M61H)
      • C5.Blank

H2O

      • B1.1 (CapD CP)
      • B2.2 (CapD CP w/ Thrombin)
      • B3.3BC (CapD NSS: lower two bands)
      • B4.3ABC (CapD NSS: All bands)
      • B5.4 (CapD CP NSS)
      • B6.5 (CapD CP NSS Foldit)
      • B7.6 (T2V)
      • B8.7 (F24H,R356K)
      • B9.8 (G79A)
      • B10.9 (M61H)
      • B11.10 (M61N)
      • B12.11 (S22Q,T59Q)
      • D1.12 (T18L,T59Q,M61N,F452W)
      • D2.13 (T20S,T59Q,M61T)
      • D3.14 (T59Q,M61Q,F452W)
      • D4.15 (T59S,M61H)
      • D5.Blank
  • Ran Mass spec to determine the characteristics of second band on gel

8/16/10

  • Left proteins out over weekend in ice bucket.
    • CapD NSS and CapD CP NSS Foldit both crashed out.
  • Regrow CapD NSS and CapD CP NSS Foldit
  • Gravity columns left out over weekend.
    • Recharged, but ask Justin if should use again.
  • Autoclaved 2x 500mL TB in 2L flasks

8/17/10

  • Plate Chlor+Kan for incubating transformed cells
  • Transform CJ236 cells for creating ssDNA of CapD NSS and CapD CP NSS Foldit tomorrow
  • Inoculate and grow up 500mL TB+Kan cultures of CapD NSS and CapD CP NSS Foldit. Induce with 500uL IPTG at OD 0.5-0.8
    • Induced at 2pm. Incubating at 18C in shakers at Schief lab.

8/18/10

  • Spin down cells
  • Add phage to ssDNA prep and expand culture

8/19/10

  • Purified and concentrated protein (One concentrated down to 400uL, other to 800uL, instruction mismatch with protocol)[Correct: 200uL]
    • New columns used. Covered with parafilm since can't find caps.
  • Chris harvested ssDNA for CapD NSS and CapD CP NSS Foldit

8/20/10

  • A280 came up with CapD - 6.1mg/mL and CapDCP - 3.1mg/mL
  • Running gel for protein concentrations
    • Started staining 2:12 pm
  • Flash freeze CapD and CapDCP proteins in 50uL aliquots

8/23/10

  • Ran fluorescence readings to determine correction coefficients for enzyme assay due to product/substrate quenching

Rows

    • 1. Product concentration at 10uM, Substrate decreasing 100uM to 0uM (Concentration cut in half each time, 12th well is 0uM)
    • 2. 5uM Product, Substrate decreasing 100uM to 0uM (Concentration cut in half each time, 12th well is 0uM)
    • 3. Substrate set at 100uM, Product decreasing 100uM to 0uM (Concentration cut in half each time, 12th well is 0uM)

8/24/10

  • Still searching for proper Michaelis-Menten profile. (Abandoned 8/23/10 attempt)
    • Original Excitation/Emission standard looks fine (490/520nm).
    • Tested 0.01 and 0.00001 mg/mL protein. First too high, second too low.
    • Tested serial dilution (half) enzyme from 0.01mg/mL decreasing across 12 wells. Substrate hold at 1uM.
    • Tested serial dilution (half) substrate 100uM decreasing across 12 wells (no blank). Hold enzyme at 0.001mg/mL.
    • Testing serial dilution (half) substrate 1uM decreasing across 12 wells (12 is blank). Hold enzyme at 0.0001mg/mL.
  • Kunkel's Mutagenesis

8/25/10

Reads

  • Calibration Curve (New Substrate)
    • Substrate start 1uM serial dilution (halves) across. Product start 0.1uM serial dilution (thirds) down. PMT High Sensitivity
  • Enzyme Assay (New Substrate)
    • 6 Rows. Two CapDCP, two CapD, two no enzyme. (0.0001mg/mL, 2.3nM)
    • First row of each L-Glu. Second row of each H2O.
    • Substrate start 1uM serial dilution (halves) across. PMT High Sensitivity.
  • Calibration Curve (Old Substrate)
    • Substrate start 100uM serial dilution (halves) across. Product start 1uM serial dilution (thirds) down. PMT High Sensitivity
  • Enzyme Assay (Old Substrate)
    • 3 rows. L-Glutamate in all rows. CapD 0.01mg/mL (200nM), CapD 0.0001mg/mL (2nM), and no enzyme (0nM). PMT High Sensitivity.

All Complete. Looks like good results across the board. Begin data analysis tomorrow.

8/26/10

  • Normalized Michaelis-Menten Data which better fit Substrate Inhibition Curves.
  • Gave Dr. Baker an update on results from the past month.

8/27/10

  • Presentation Slides

8/30/10

  • Dilute and Kinase Oligos
    • A1. CapDCPNF I83T
    • A2. CapDCPNF I83V
    • A3. CapDCPNF T2C
    • A4. CapDCPNF T2S
    • A5. CapDCPNF T18S_T20S
    • A6. L40A
    • A7. M61A
    • A8. M61C
    • A9. M61D
    • A10. M61G
    • A11. M61I
    • A12. M61L
    • B1. M61N
    • B2. M61S
    • B3. M61T
    • B4. M61V
    • B5. T20S_F24H
    • B6. T20S_F24Y
    • B7. Control
    • B8. Tossed
    • B9. T20S
    • B10. T59N
    • B11. T59S_M61S


  • Pipetted 3uL of 8-17 kinased oligos into eppendorf tube marked M61X

Overnights Growing at 37C

  • 1.CapDCPNFDelta
  • 2.F24A
  • 3.F24H
  • 4.F24H,T59N
  • 5.F24Y,R305K
  • 6.F24Y,T59N
  • 7.F24Y,T59N,R305K
  • 8.L40R
  • 9.L40S
  • 10.L40W
  • 11.M61S
  • 12.N23K
  • 13.N23Q
  • 14.T20A
  • 15.T20C
  • 16.T20S,T59N
  • 17.T59M
  • 18.T59N
  • 19.T59S
  • 20.CapD CP NSS Foldit (Control)

8/31/10

  • Kunkel's up to plate and incubate cells
    • 1.I83T
    • 2.I83V
    • 3.L40A
    • 4.T2C
    • 5.T2S
    • 6.T2S,T18S_T20S
    • 7.T2S,T18S_T20S,T59S_M61S
    • 8.T2S,T20S
    • 9.T18S_T20S
    • 10.T20S_F24H
    • 11.T20S_F24Y
    • 12.T20S_F24Y,T59N
    • 13.T20S,M61A
    • 14.T20S,M61C
    • 15.T20S,M61D
    • 16.T20S,M61G
    • 17.T20S,M61I
    • 18.T20S,M61L
    • 19.T20S,M61N
    • 20.T20S,M61S
    • 21.T20S,M61T
    • 22.T20S,M61V
    • 23.T59S_M61S
    • 24.Control (Blank)
    • 25.Control (Only Cells)

There is an extra plate named 22,23?. Numbering may have been skewed somewhere so one plate should end up with no colonies since nothing was put on it.

Grow up proteins(small scale) to OD600 ~0.4 and induce with 25uL IPTG.

  • 1.CapDCPNFDelta
  • 2.F24A
  • 3.F24H
  • 4.F24H,T59N
  • 5.F24Y,R305K
  • 6.F24Y,T59N
  • 7.F24Y,T59N,R305K
  • 8.L40R
  • 9.L40S
  • 10.L40W
  • 11.M61S
  • 12.N23K
  • 13.N23Q
  • 14.T20A
  • 15.T20C
  • 16.T20S,T59N
  • 17.T59M
  • 18.T59N
  • 19.T59S
  • 20.CapD CP NSS Foldit (Control)

9/1/10

Have not tested F24W, R305A

Overnight: Kunkel's Plate shake in warm room

  • 1.I83T: A1-D1
  • 2.I83V: E1-H1
  • 3.L40A: A2-D2
  • 4.T2C: E2-H2
  • 5.T2S: A3-D3
  • 6.T2S,T18S_T20S: E3-H3
  • 7.T2S,T18S_T20S,T59S_M61S: A4-D4, E12-F12
  • 8.T2S,T20S: E4-H4
  • 9.T18S_T20S: A5-D5
  • 10.T20S_F24H: E5-H5
  • 11.T20S_F24Y: A6-D6
  • 12.T20S_F24Y,T59N: E6-H6
  • 13.T20S,M61A: A7-D7
  • 14.T20S,M61C: E7-H7
  • 15.T20S,M61D: A8-D8
  • 16.T20S,M61G: E8-H8
  • 17.T20S,M61I: A9-D9
  • 18.T20S,M61L: E9-H9
  • 19.T20S,M61N: A10-D10
  • 20.T20S,M61S: E10-H10
  • 21.T20S,M61T: A11-D11
  • 22.T20S,M61V: E11-H11
  • 23.T59S_M61S: A12-D12
  • 24.Blank: E12-H12

Spin Down Cells at 4:00pm. Place in -20C

  • 1.CapDCPNFDelta
  • 2.F24A
  • 3.F24H
  • 4.F24H,T59N
  • 5.F24Y,R305K
  • 6.F24Y,T59N
  • 7.F24Y,T59N,R305K
  • 8.L40R
  • 9.L40S
  • 10.L40W
  • 11.M61S
  • 12.N23K
  • 13.N23Q
  • 14.T20A
  • 15.T20C
  • 16.T20S,T59N
  • 17.T59M
  • 18.T59N
  • 19.T59S
  • 20.CapD CP NSS Foldit (Control)


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