"
Team:Georgia State/Notebook
From 2010.igem.org
July 1, 2010
Joe
Plated transformed cells containing either GFP (control) or iGEM part onto LB plates with 10µg/mL ampicillin (2 plates each, 10mL or 100mL) Also plated ≈100mL (remaining amount) of cells onto LB plates not containing ampicillin. Spread plates with hockey stick and placed in 37°C at 7:35.
July 2, 2010
Alykhan and Virginia DNA purification and ligation Replated E. coli culture
July 6, 2010
Dan
Replated E. coli culture
Alykhan
Transformation 9A, 8I (digested) & GFP.
July 7, 2010
Joe, Kendra, Angie
Replated colonies from 9A RFP plate (3 plates). Colonies on broth 100 µL for 9A but no fluorescence observed 9A survivor colonies replated onto 10 µg/mL ampicillin plates and incubated at 30°C Mother plate in fridge Made 1000mL YPD Growing P. anomala in YPD broth for competent cells protocol
July 8, 2010
Angie, Kendra, Melissa, Nishedh, Alykhan
Diluted pichia cells in a volume of 50mL to a OD600 = .186+.201 Transform pars 12E and 12M into E. coli
July 13, 2010
Virginia 10 glycerol stocks of each
o P. pastoris
o 9A E. coli
Inoculated 12E broth for plasmid extract and glycerol stocks Plate of P. pastoris for quality control
July 14, 2010
Virginia, Joe, Angie, Alykhan
10 glycerol stocks of 12E P. pastoris competent cells Started, ready at 2pm Plasmid resuspension buffer made LB agar aliquots
o warm in water bath
o one 10mL tube for 1L agar
extraction of 12E+9A plasmid parts: white tubes in freezer
July 15, 2010
Virginia, Angie, Alykhan, Joe
Cut plasmid and ligated P. pastoris cells ready in -80°C
July 20, 2010
Melissa, Alykhan
Prepared tris HCl for buffer solutions
July 22, 2010
Virginia
Prep LB, YPD plate solution Prep NEB 2+3 buffer solutions
August 3, 2010
Joe, Virginia
Tried to extract 12E plasmid with Jetgene kit starting with cells stored in resuspension buffer. (Started with step 2). Cell solution was very viscous. Difficult to pipette. Did not form a pellet in step 4. Inoculated LB+Ampicillin with 12E and stored at 37°C from glycerol stock in -80°C.
o Will try again with fresh cells
o Ran out of buffer in Jetgene kit.
August 4, 2010
Virginia 12E plasmid extraction from 5-min kit. Lysis solution and wash buffer taken from eppendorf kit needs to be confirmed on Nano Drop.
August 5, 2010
12E SpeI protocol, PstI protocol 9A PstI proocol 9A was purified as the Xba I previously used on it was not fast digest Gel extraction of 9A and 12E
August 8, 2010
Joe
Prepared YPD+sorbitol
o 10g yeast extract
o 20g peptone
o 182.2g sorbitol
o 900ml H20
Inoculated 250ml flash of YPD with P. pastoris from 6-29-10 culture in SAB
o added 500ml of SAB culture into ≈100-125ml YPD, stored in shaker in 30°C incubator
August 8, 2010
Centrifuge P. pastoris to prep for electroporation Competent cells prepared, ready to pulse Transformed/electroporated P. pastoris with 9A and GFP
August 9, 2010
Joe
Prepared YPD/Hepes Prepared 10mL of DTT Inoculated 2 1L flasks containing 250mL YPD with 500mL of P. pastoris from overnight culture. Stored in -80°C room at 30°C in shaker. Protocol modifications for P. pastoris transformation are in notebook
August 12, 2010
Reading about P. pastoris as vaccine vector
Started engineering of plasmid
Joe
Inoculated LB+Ampicillin broth with glycerol stock of E. coli containing GFP
Located in 37°C incubator in Crow lab.
September 2nd, 2010
Melissa, Joe
Inoculated LB broth with 250 uL 12E 100 mL freshly-made LB broth + 1 mL LB + 10x ampicillin ~100 mL 1x ampicillin + LB broth 12E placed in shake flask @ 37°C
September 7th, 2010
Alykhan, Nishant, Bhavik, Kathy, Nishedh Innoculated 12E (arabinose-induced promoter) Plates put in 37°C incubator for 14 hours P. pastoris from -80°C inoculated in LN broth and plated. Grown for 2 days. DNA extracted and placed in -20°C freezer [PCR needs to be done using primers] 6G (Lac regulating promoter) was transformed into E. coli Competent cells were used from -20°C freezer Remainder of DNA was placed in -20°C freezer Transformation was done in duplicate and plates were put in 37°C incubator for 14 hours Growing part was transformed to broth and a new plate [parts must be ligated; DNA must be extracted]
September 10th, 2010
Joe Prepared 1x working stock of Amro’s Cox 5-R (Ref# 49115848) and Cox 5-F (Ref# 49115847) to use as positive control for PCR reactions 90 µL ddH2O + 10 µL 10x stock = 100 µL 1x working stock
September 13th, 2010
Joe “Isolation of Histidinol Dehydrogenase from Pichia pastoris using PCR” Two sets of primers were designed based on previously published primers but modified to add EcorI and SpeI sites Set I: F=0848 R=0849 Set 2: F=0850 R=0851 Cont: F=Cox5 R=Cox5 Used following ratios: 2.5 µL MasterMix, 1 µL F. primer, 1 µL R. primer, 1 µL DNA (45 mg/mL), 9.5 µL water. Total volume 25 µL in PCR reaction tubes. PCR settings: adjusted 74 time from 30 sec → 3:00 min Reaction: F R Control Cox5 Cox5 Set 1 0848 0841 Set 2 0850 0851 Hybrid 1 0848 0851 Hybrid 2 0850 0849
Tube labels: Con, 1, 2, 3, 4. Started at 3:00 PM, finished at 5:35 PM.
September 21st, 2010
“Insertion of His4 PCR product into plasmid” Purpose: to insert the PCR produce of F=0850 and R=0851 into the RFP plasmid Procedure: (PCR) 9A (RFP) digest with SpeI and EcoRI: 15 µL nuclease-free H20, 2 µL fast digest 10x buffer, 2 µL DNA, 1 µL each enzyme. Total volume=21 µL H (histidinol) digest with SpeI and EcoRI: 17 µL nuclease-free H20, 2 µL fast digest, 10 µL DNA, 1 µL each enzyme. Total volume=31 µL Heated at 37°C 5 min Purification of plasmid and His4: used QIA quick PCR purification protocol Plasmid: Total volume from RE digest = 20 µL. 20 µL plasmid + 100 µL PBI buffer (5x PBI) = 120 µL H: total volume = 30 µL. 30 µL + 150 µL PBI buffer Gel: _______ _______ _______ _______ _______ _______ _______ L 9A _______ P L A 9A U A D C D D D E E R R
Extracted and plated with LB on agar. Poor plates. Discussion: This experiment failed at some point as noted by the lack of bands after running a gel to isolate the 9A plasmid backbone. The successful banding patterns by Kristin’s PUC plasmid indicate that the error occurred in either the restriction digest or during the purification process. It is most likely that the problem was in the restriction digest. The enzymes used were fast-digest enzymes and were not inactivated after the 5 min incubation. They were placed at room temperature for approximately an hour before being purified. It is believed that the enzymes continued to digest the DNA to completion. Future attempts should require a quicker transition time from the digest, an inactivation step, and potential postponing of the purification step until after the ligation has been performed.
September 22nd, 2010
“PCR with primers 0850 and 0851” The purpose is to obtain His4 fragment from P. pastoris DNA. This reaction was performed successfully on September 10th , 2010. P. pastoris DNA 580 ng/µL. 1:10 dilution → 58 ng 12.5 µL MasterMix 2x, 1 µL F. primer, 1 µL R. primer, 1 µL DNA (58 ng/µL), 9.5 µL H2O)
September 22nd, 2010
“Insertion fo His4 PCR product into 9A plasmid backbone” The purpose is to ligate PCR product of F=0850 and R=0851 into RFP (9A) plasmic backbone. The process involves digesting both components with EcoRI and SpeI, running a gel to remove RFP, and ligation. Procedure: 1. Digest a. . 9A digest: 15 µL nuclease-free H2O, 2 µL fast-digest 10x buffer, 2 µL DNA, 1 µL of each enzyme. Total volume is 21 µL. b. His4 digest (PCR fragment): 17 µL nuclease-free H2O, 2 µL fast-digest 10x buffer, 10 µL S3 (his4), 1 µL each enzyme. Total volume is 31 µL. Inactivate at 80°C for 5 min in thermocycler 2. Purification of His4 fragment a. Add 5 volumes of buffer PBI to 1 volume sample i. 150 µL PBI to 30 µL sample b. Place into a QIA quick column inside a 2 mL collection tube c. Centrifuge 60 s and discard flowthrough d. Add .75 mL PE to column i. Centrifuge 60 s and discard flowthrough e. Centrifuge again for 6 seconds f. Place column in clean centrifuge, add 50 µL EB buffer, and spin 60 s to elute DNA g. Place on ice in centrifuge tube
3. Gel excision of 9A plasmid backbone a. Also ran PCR products from 0850 and 0851 from today and 9-10-10 C RFP H1 H2 ______ ______ ______ ______ ______ ______ ______ ______ L L A ______ A D ______ ______ D D D E ______ E R ______ R
Cut out 3 gels segments: RFP, backbone, H.S. Weights: 0.30 g plasmid backbone, 0.15 g RFP, 0.40 g His4 Add 3 volumes of Buffer QG to 1 volume of gel (100 mg~100 µg) Plasmid backbone = 300 mg → 900 µL QG RFP = 150 mg → 450 µL QG His4 = 400 mg → 1200 µL Incubate for 10 min @ 50°C. Shake periodically to help dissolve gel. Add one part isopropanol and mix PB → +300 µL RFP → +150 µL His4 → +400 µL Apply to spin column (800 µL of sample at a time). Centrifuge for 1 min. Add .5 mL QG to column and spin 1 min Wash with .75 mL PE. Centrifuge 1 min. Discard flow-through. Spin again for 1 min. Elute DNA by placing column into clean centrifuge tube and applying 50 µL EB buffer
September 23rd, 2010
9A backbone with sticky ends. Ligate with His4 after digestion (EcoRI and SpeI) His4 → 11.7 ng/ µL Plasmid backbone → 8.3 ng/µL Ligation: used 3 µL of 8 ng/µL plasmid backbone → 24 ng; and 41 µL of 11 ng/ µL His4 → 44 ng Incubated with thermal cucler for 4 hours at 16°C, 10 min @ 70°C, and hold at 4°C overnight Ligation: Used 1 µL 10x ligase bugger, 0.5 µL mM DDT, 1 µL 10 mM ATP, 3 mL plasmid backbone, 10 µL ligase dilution buffer, 4 µL His4
September 23rd, 2010
“Isolation of pAOX1 and pGAP” A → pAOX1 B→ pGAP C→Control (cytochrome from Dr. Amro) Master Mix 12.5 µL Forward primer 1 µL Reverse primer 1 µL Pichia DNA 1 µL Water 9.5 µL
September 23rd, 2010
Bianca Ligation Add these in a PCR tube: 1. 1 µL 10x ligase buffer 2. 0.5 µL 100 mM DDT 3. 1 µL 10 mM ATP 4. 3 µL plasmid backbone 5. 1 µL DNA ligase 6. 4 µL His4 Total volume is 10 µL 1 µL (0.1 TH DNA ligase + 0.9 DNA ligase buffer) In thermocycler @ 6:26
September 27th, 2010
Two LH and two LR lanes run at 100 volts, 400 mA, 60 minutes. Gel: 2.5 µL brotium gel red, 0.43 g agarose, 35 mL TAE
October 2nd, 2010
Alykhan The ligated RFP and plasmid backbone and the part (His-4) were run in a gel to check the ligation. 1,2% agarose gel was made (0.9 g in 75 mL). 7.5 µL of biotine red was added Lane 1: ladder; 2: RFP; 3: RFP; 4: Part 1; 5: Part 2; 6: ladder 10 µL of sample and 2 µL of formaldehyde dye was loaded. 6 µL of ladder was loaded. Gel was run for one hour at 100 volts Excision: 1st: 0.78 g → 0.89 g 0.11 (110 mg) 2nd: 0.80 → 0.88 80 mg Add buffer: 330 µL to 1st and 80 µL to 2nd After dissolves: add 110 µL isopropenol Sample 1: 7.1 Sample 2: 7.4
October 4th, 2010
Joe “Isolation of pGAP, pAOX1, FLD1” The purpose of this experiment is to isolate the parts pGAP, pAOX1, and FLD1 from DNA of P. pastoris through PCR rxn. 2 sets of primers have been designed for each part. The first set of each contains an Xba site on the forward primer and a Spe1 site on the reverse. The second set of primers adds EcoRI, Not1, and Xba to the forward and Spe1, Not !, and Pst onto the reverse primer. The second product of the second set will yield a biobrick product, ready for insertion into plasmid _______. However, because adding 3 restriction enzyme sties decreases primer quality, hybrid reactions will be run to increase likelihood of obtaining a useable product. Methods Tube # FLD1 F1 (F) 8286 (R) 8287 F2 (F) 8286 (R) 8289 F3 (F) 8288 (R) 8287 F4 (F) 8288 (R) 8289 Product: X—S, X—SNP, ENX---S, ENX---SNP → high temp pGAP G1 (F) 1148 (R) 1149 G2 (F) 1148 (R) 8285 G3 (F) 8284 (R) 1149 G4 (F) 8284 (R) 8285 X---S, X---SNP, ENX---S, ENX---SNP → high temp pAOX1 A1 (F) 1146 (R) 1147 A2 (F) 1146 (R) 8283 A3 (F) 8282 (R) 1147 A4 (F) 8282 (R) 8283 X---S, X---SNP, ENX---S, SNX---SNP → high temp For each reaction, the following ratio was used: 12.5 µL MasterMix, 1 µL forward primer, 1 µL reverse primer, 1 µL 58 ng/µL Pichia DNA, 9.5 µL H2O. 25 µL total. [ran gel] F: 1.3 Kb (contains EcoRI site! Must use different enzyme!) A: .9 Kb (Gel showed successful amplification of all samples except F1) G: .5 Kb For isolating pGAP, the FR sample showed the strongest bands. However, it was inaccessible from extraction.
October 5th, 2010
Nishedh and Bhavik Restriction digest of the parts. Cut the A4, G4 with EcoRI and PstI. Reaction volumes: A4: 10 µL water, 2 µL 10x buffer, 17 µL DNA, 1 µL EcoRI, 1 µL Pst1. Total 31 µL. 5 min at 37 degrees C. G4: 17 µL water, 2 µL 10x buffer, 10 µL DNA, 1 µL EcoRI, 1 µL PstI. Total 31 µL. 5 min at 70 degrees C Since F4 has an EcoRI site in it, we will use a different approach. Cut F4 with X-ba1 and PstI First we have to cut both with Xba1: Reaction volume: F4: 10 µL DNA, 18 µL water, 2 µL 10x buffer, 2 µL Xba1 = 32 µL. RFP: 5 µL DNA, 23 µL water, 2 µL 10x, 2 µL Xba1: 32 µL. Overnight in the thermocycler. Purification of A4, G 31 µL x 5 = 155 µL PBI spin 750 µL PE. Spin. Spin again. 50 µL of EB spoin. Quantify A4 → 9.5 ng/µL G4 → 5.7 ng/ µL Ligation Mix all these together in PCR tubes: 1 µL 10x ligase buffer, 0.5 µL 100 mM DDT, 1 µL of 10 mM ATP, 1 µL plasmid vector PCBIA3, 10 µL DNA, 0.9 µL ligase dilution buffer, 0.1 µL T4 DNA ligase, 0.5 µL nuclease-free water. Total: 15 µL. Put in thermocycler. Purify F & R 20 mins. Fast digest with PstI 10 min. Ligate F4 and R in vector.
Isolation of pTEF from PCR The purpose of this experiment is to isolate the pTEF promoter from pichia pastoris DNA. Two sets of primers were used for this experiment to produce PCR products with different restriction sites. The primers used were stored in the -20° C freezer. Set 1 Sites Added Forward 3414 X---- Reverse 3415 ----S
Set 2 Sites Added Forward 9201 ENX--- Reverse 9202 ---SNP
PCR 12.5 µL Mastermix 1 µL Forward 1 µL Reverse 1 µL Protein DNA (58 ng/µL) 9.5 µL double sterile water PCR Rxn Tube # Forward Primer Reverse Primer Product 1 3414 3415 X----S 2 3414 9202 X----SNP 3 9201 3415 ENX----S 4 9201 9201 ENX----SNP
Modifications: - Samples 1-3 were modified from 55°C →57°C. - Sample 4: run on IGEM high temp cycle. Modified from 66°C→63°C.
How to Prepare a Gel
Need: 1.2% agarose and .001 % Bio-red Examples 35 ml gel → 0.42 g Agarose 35 ml 1X TAE 3.5 µL Bio-Red Or 75 ml gel → 0.9 g Agarose 75 ml 1X TAE 7.5 µL H₂O 1. Add weighed amount of agarose to flask 2. Add 1X TAE 3. Microwave for ~60 seconds, stirring occasionally 4. Prepare gel tray with desired wells in place 5. Add Bio-Red after microwaving, swirl to mix, and pour into gel tray. 6. Let solidify for at least 30 minutes. 7. Remove wells.
Gel Electroporation A₄ & G₄ → The ligated parts were ran in a gel to check if the ligation was successful. The purified % digested F₄ and RFP were ran on a gel to check if they were successful. Ladder F₄ A₄ G₄ RFP Ladder - None - - - - - - - -
Ran at 120 V, 200 mA, for 60 minutes. Results: Two bands were located in the RFP lane Indicates: 1- RFP (part) 2- Plasmid backbone. A₄ & G₄- -1 band in each lane around 3000 base pairs. This could possibly indicate that the past was ligated into the plasmid backbone.
Transformation A₄ &G₄ were transformed using the heat shock protocol. Results: - No growth on LB with ampicillin plates for either part. - LB plates displayed viability. - Primers needed to be made to amplify the ligated part because the gel reveals evidence of ligation, but transformation not successful.
October 18th, 2010
“Transformation of TOP10 cells” Melissa TOP10 cells were transformed (TOP10 protocol) with alcohol oxidase, glyceraldehyde-3-phosphate, and RFP as a control. Goal is to determine optimal media for growth. 2 µL of each were added to 50 µL vials of competent cells. Incubated on ice 30 min. Heat shock at 42 degrees C 30 sec. 250 µL SOC beroth added to each. Vials centrifuged at 37 degrees 1 hour. Plated. 20 µL each on LB plated with ampicillin. 700 µL each on LB plated. 100 µL each on YPD as control. Plates inverted and incubated at 37 degrees 14 hours.
Gel electrophoresis: 90 A4 – 0.09: 0.80 – 0.89 (6.0) 220 F4 – 02.2: 0.75 – 0.98 (9.3) 230 G2 – 0.23: 0.82 – 1.05 (9.4) 300 T1 – 0.30: 0.81 – 1.05 (9.0) QG A4 → 270 µL F4 → 660 µL G2 → 690 µL T1 → 900 µL
October 20th, 2010
Angie Digestion: G1 and F digest with Pst1 17 µL DNA, 10 µL water, 2 µL 10x buffer, 1 µL Pst1 = 30 µL Purify using PCR purification kit.
