Team:Columbia-Cooper-NYC/Columbia notebook 2
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<a href="https://2012.igem.org/Team:Columbia-Cooper-NYC/Outreach">Outreach</a> | <a href="https://2012.igem.org/Team:Columbia-Cooper-NYC/Outreach">Outreach</a> | ||
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<a href="https://2012.igem.org/Team:Columbia-Cooper-NYC/Safety">Safety</a> | <a href="https://2012.igem.org/Team:Columbia-Cooper-NYC/Safety">Safety</a> | ||
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= Columbia Genetics Lab Notebook = | = Columbia Genetics Lab Notebook = | ||
+ | __NOTOC__ | ||
+ | <div class="content-header-box"> | ||
== July, 2012 == | == July, 2012 == | ||
+ | </div> | ||
+ | <div class="content-body-box"> | ||
==== Thursday, 5th ==== | ==== Thursday, 5th ==== | ||
* Re-hydrated plasmids with 50µl of LB and Kanamycin solution | * Re-hydrated plasmids with 50µl of LB and Kanamycin solution | ||
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*#ho1 | *#ho1 | ||
* Isolated ho1 and PcyA using standard plasmid isolation protocol | * Isolated ho1 and PcyA using standard plasmid isolation protocol | ||
+ | |||
+ | </div> | ||
== August, 2012 == | == August, 2012 == | ||
+ | <div class="content-body-box"> | ||
==== Wednesday, 1st ==== | ==== Wednesday, 1st ==== | ||
* Applied IPTG to samples of bacteria with GFP or kill gene and placed back in incubator at 37C | * Applied IPTG to samples of bacteria with GFP or kill gene and placed back in incubator at 37C | ||
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* Ran gel for 25 minutes at constant 150V | * Ran gel for 25 minutes at constant 150V | ||
* Took picture under UV light | * Took picture under UV light | ||
+ | ==== Wednesday, 22nd ==== | ||
+ | * Conducted ligations for the following using a vector to insert ratio of 1:3 | ||
+ | *# Sample 1: Inducible promoter (upstream insert)-Kill gene (Downstream insert)-pSB3C5 (vector) | ||
+ | *# Sample 2: Inducible promoter (upstream insert)-Upps 5 (Downstream insert)-pSB3C5 (vector) | ||
+ | *# Sample 3: Inducible promoter (upstream insert)-Upps 6 (Downstream insert)-pSB3C5 (vector) | ||
+ | *# Sample 4: Upps 4 (upstream insert)-Kill gene (Downstream insert)-pSB1T3 (vector) | ||
+ | *# Sample 5: Upps 4 (upstream insert)-Upps 5 (Downstream insert)-pSB1T3 (vector) | ||
+ | *# Sample 6: Upps 4 (upstream insert)-Upps 6 (Downstream insert)-pSB1T3 (vector) | ||
+ | *# Sample 7: Control pSB3C5 (1:0 vector to insert ratio) | ||
+ | *# Sample 8: Control pSB1T3 (1:0 vector to insert ratio) | ||
+ | ''Note: above ligations will be done with T4 buffer and ligase from both the Biobrick kit and Columbia's lab'' | ||
+ | |||
+ | ''Note 2: label all sample using iGEM ligase and buffer with prefix N'' | ||
+ | |||
+ | ''Note 3: label all sample using Columbia lab's ligase and buffer with B'' | ||
+ | * Electroporated the following using the standard protocol provided by Bioline: | ||
+ | *# Constitutive promoter-BBa_J130002 | ||
+ | *# Low-copy TET plasmid-PSB3T5 | ||
+ | *# High-copy CAM plasmid-PSB1C3 | ||
+ | *# High-copy Amp/CAM plasmid-PSB1AC3 | ||
+ | *# High-copy Amp/TET plasmid-PSB1AT3 | ||
+ | *# PUC19 control plasmid | ||
+ | * Chemically transformed the following using the heat shock protocol: | ||
+ | *# N1, N2, N3, N7, B1, B2, B3, B7 into BL21 (DE) pLysZ orange cells | ||
+ | *# GFP IPTG given by graduate student at Columbia into BL21 (DE) green cells | ||
+ | *# PUC19 into BL21 (DE) pLysZ orange cells | ||
+ | *# PUC19 into BL21 (DE) green cells | ||
+ | * Purified ligated protocol for electroporation following QIAquick PCR purification protocol | ||
+ | * Plated all chemically transformed and electroporated cells | ||
+ | |||
+ | ==== Thursday, 23rd ==== | ||
+ | ''Note: Observed no growth with pSB1T3, BL21 pLyse'' | ||
+ | |||
+ | ''Note 2: Observed growth with PUC19 and other BL21'' | ||
+ | * Streaked sample N5 | ||
+ | * Found following 3 restriction sites (to be double checked by graduate student): | ||
+ | *# NgomIV: CCGCCGGC | ||
+ | *# EcoRI: GAATTC | ||
+ | *# PstI: CTGCAG | ||
+ | ==== Friday, 24th ==== | ||
+ | * Diluted IPTG driven cultures to 200x with fresh LB/antibiotic solution | ||
+ | * Followed standard plasmid isolation protocol for everything except the following: | ||
+ | *# 1 sample of graduate student's GFP | ||
+ | *# Sample of pSB1C3 (the high copy CAM plasmid) | ||
+ | * Checked optical densities of samples B1-B3 and graduate student's GFP | ||
+ | * Created two subsamples each of B1-B3; applied 1M IPTG to half of the subsamples | ||
+ | * Applied 1M IPTG to into one of the samples of graduate student's IPTG-GFP | ||
+ | ==== Saturday, 25th ==== | ||
+ | * Checked IPTG "induced" samples and observed no expression | ||
+ | * Measured optical density for following samples and diluted the samples: | ||
+ | *# GFP by graduate student-pre-dilution: 2.0, post dilution: 0.710 | ||
+ | *# IPTG-kill (N1)-pre-dilution: 2.4, post dilution: 0.736 | ||
+ | *# IPTG-U5 (N2)-pre-dilution: 1.9, post dilution: 0.740 | ||
+ | *# IPTG-U6 (N5)-pre-dilution: 2.4, post dilution: 0.630 | ||
+ | *# IPTG-U5 (B2)-pre-dilution: 1.9, post dilution: 0.681 | ||
+ | *# IPTG-U6 (B3)-pre-dilution: 1.9, post dilution: 0.651 | ||
+ | * Applied 1.5μL of 1M IPTG with 1.5mL of each sample and place in 25C shaker | ||
+ | * Followed standard plasmid isolation protocol for N1 and N5 | ||
+ | ==== Monday, 27th ==== | ||
+ | * Observe expression of graduate student's GFP | ||
+ | * Measured optical densities for following: | ||
+ | ** Ligated samples: | ||
+ | **# N1-N3 | ||
+ | **# B1-B3 | ||
+ | ** Mini-prepped: | ||
+ | **# Upps1-Upps6 | ||
+ | **# pSB1C3 | ||
+ | **# pSB1T3 | ||
+ | **# pSB1AT3 | ||
+ | **# pSB1AC3 | ||
+ | **# pSB1A2 | ||
+ | * Sent above samples for sequencing | ||
+ | * Followed protocol from bioline for digestion: | ||
+ | *# Downstream: Upps 1 | ||
+ | *# Upstream: Constitutive promoter | ||
+ | *# Destination plasmid: pSB3T5 | ||
+ | *# Destination plasmid: pSB1C3 | ||
+ | *# Destination plasmid: pSB1AC3 | ||
+ | *# Destination plasmid: pSB1AT3 | ||
+ | * Streaked out Upps 1 from agar stab | ||
+ | |||
+ | ==== Tuesday, 28th ==== | ||
+ | * Reviewed results from the sequencing from Genewiz | ||
+ | * Design and send new primer (nev) for sequencing | ||
+ | * Conducted ligations for the following using a vector to insert ratio of 1:3 | ||
+ | *# Constitutive promoter (upstream insert)-Upps 1 (downstream insert)-pSB1C3 (vector) | ||
+ | *# Constitutive promoter (upstream insert)-Upps 1 (downstream insert)-pSB3T5 (vector) | ||
+ | *# Constitutive promoter (upstream insert)-Upps 1 (downstream insert)-pSB1AT3 (vector) | ||
+ | *# Constitutive promoter (upstream insert)-Upps 1 (downstream insert)-pSB1AC3 (vector) | ||
+ | * Electroporated the above 4 ligations without purifying the ligations | ||
+ | * Plated cells into respective plates | ||
+ | * Picked colonies for Upps 1-YF1/FixJ | ||
+ | ==== Wednesday, 29th ==== | ||
+ | * Conducted ligations for the following using a vector to insert ratio of 1:3 | ||
+ | *# Upps 4 (upstream insert)-Upps 5 (downstream insert)-pSB1C3 (vector) | ||
+ | *# Upps 4 (upstream insert)-Upps 6 (downstream insert)-pSB1C3 (vector) | ||
+ | *# Upps 4 (upstream insert)-Kill gene (downstream insert)-pSB1C3 (vector) | ||
+ | *# Upps 4 (upstream insert)-Upps 5 (downstream insert)-pSB1AC3 (vector) | ||
+ | *# Upps 4 (upstream insert)-Upps 6 (downstream insert)-pSB1AC3 (vector) | ||
+ | *# Upps 4 (upstream insert)-Kill gene (downstream insert)-pSB1AC3 (vector) | ||
+ | * Extracted Upps 1 (Kan) from glycerol stock from -80C freezer | ||
+ | * Followed graduate student's quickchange protocol and labelled N1-N4, E1-E4, P1-P4 with the labeling as following: | ||
+ | *# Sample 1: HF only | ||
+ | *# Sample 2: HF and 1.5μL DMSO | ||
+ | *# Sample 3: GC only | ||
+ | *# Sample 4: GC and 1.5μL DMSO | ||
+ | *# Prefix N: NgoMIV mutation | ||
+ | *# Prefix E: EcoRI mutation | ||
+ | *# Prefix P: PsH mutation | ||
+ | ==== Thursday, 30th ==== | ||
+ | * Followed standard plasmid isolation protocol for Upps 1 | ||
+ | * Transformed U4-U5, U4-U6, U4-kill in 2 different plasmids | ||
+ | * Ran gels of Quickchange product | ||
+ | *# Column 1: miniprepped plasmid | ||
+ | *# Column 2-5: N1-N4 | ||
+ | *# Column 6-9: E1-E4 | ||
+ | *# Column 10-13: P1-P4 | ||
+ | * Re-ligated Constitutive promoter-Upps 1 | ||
+ | |||
+ | </div> | ||
+ | == September, 2012 == | ||
+ | <div class="content-body-box"> | ||
+ | ==== Wednesday, 5th ==== | ||
+ | ''Note: All work done from this day on was done at the Kanbar lab at the Cooper Union'' | ||
+ | * Single digested the J13002 | ||
+ | * Placed the digest above and other digests to be run on gels into -20C freezer | ||
+ | * Made 4 50mL 1% agarose gels | ||
+ | * Followed standard plasmid isolation protocol for kill gene | ||
+ | ==== Friday, 7th ==== | ||
+ | * Ran two gels to check digests | ||
+ | ** Gel Number 1 | ||
+ | **# Marker | ||
+ | **# Upps 1 | ||
+ | **# Inducible promoter 1 | ||
+ | **# Inducible promoter 2 | ||
+ | **# pSB3T5 | ||
+ | **# pSB1C3 | ||
+ | **# Low Copy Vector 1 | ||
+ | **# Low Copy Vector 2 | ||
+ | ** Gel Number 2 | ||
+ | **# Marker | ||
+ | **# Kill gene | ||
+ | **# PcyA | ||
+ | **# TetR 1 | ||
+ | **# TetR 2 | ||
+ | **# GFP from biobrick kit | ||
+ | **# GFP from Tushar | ||
+ | **# GFP from Kevin | ||
+ | * Conducted digestions for the following: | ||
+ | *# Inducible promoter digested with EcoRI&Spe with Buffer E and Buffer Multi (for 30 min.) | ||
+ | *# TetR digested with EcoRI with Buffer H | ||
+ | *# TetR digested with Spe with Buffer B | ||
+ | *# Upps 1 digested with Xba&Pst with Buffer H | ||
+ | *# Kill gene digested with Xba&Pst with Buffer H | ||
+ | *# GFP digested with Xba&Pst with Buffer H | ||
+ | *# pSB3T5 with EcoRI&Pst with Buffer H | ||
+ | *# Low copy plasmid 2 digested with EcoRI&Pst with Buffer H | ||
+ | *# pSB1C3 digested with EcoR&Pst with Buffer H | ||
+ | ''Note: Digests 7-9 was done in Antartic Phosphotase Treat 30 min in 37C and 15 min in 65C'' | ||
+ | * Conducted ligations for the following (followed by chemical transformations: | ||
+ | *# Upstream: Inducible promoter; Downstream: Upps 1; Destination plasmid: Low copy plasmid 2 | ||
+ | *# Upstream: Inducible promoter; Downstream: Kill; Destination plasmid: Low copy plasmid 2 | ||
+ | *# Upstream: Inducible promoter; Downstream: GFP; Destination plasmid: Low copy plasmid 2 | ||
+ | * Conducted additional chemical transformations with the following vectors: | ||
+ | *# pET20b | ||
+ | *# pET26b | ||
+ | *# pUC19 | ||
+ | * Conducted following additional ligations, reference numbers correlate to numbers shown above for this day's digestions | ||
+ | *# Label A: 2 and 9 | ||
+ | *# Label B: 3 and 4 and 9 | ||
+ | *# Label C: 1 and 4 and 8 | ||
+ | *# Label D: 1 and 5 and 8 | ||
+ | *# Label E: 1 and 6 and 8 | ||
+ | *# Label F: 3 and 4 | ||
+ | *# Label G: 3 and 5 | ||
+ | *# Label H: 3 and 6 | ||
+ | |||
+ | ==== Tuesday, 11th ==== | ||
+ | * Observed the following: | ||
+ | *# Inducible promoter-GFP-Low Copy CAM Plasmid had 3 pink clones (IPTG applied) | ||
+ | *# Inducible promoter-GFP-Low Copy CAM Plasmid had 3 pink clones (NO IPTG) | ||
+ | *# Inducible promoter-Upps 1-Low Copy CAM Plasmid had 32 pink clones | ||
+ | *# Inducible promoter-Kill gene-Low Copy CAM Plasmid had 50 pink clones | ||
+ | ''Note 1: Destination plasmid/vector contained J04450 biobrick part, explained pink clones'' | ||
+ | |||
+ | ''Note 2: All vectors were all treated with Antartic Phophotase, expect no religation of J04450 with LC vector'' | ||
+ | * Digested Kill gene and GFP with E and P | ||
+ | * AP Treat Kill gene with X and P, GFP with X and P | ||
+ | * Conducted ligations for the following: | ||
+ | *# GFP (E and P) with pSB1C3 (E and P, AP treated) | ||
+ | *# GFP (X and D, AP treated) with TetR/s | ||
+ | * Condcuted transformations for the following: | ||
+ | *# Ligation F from 9/7, TetR, Upps 1(X and P) | ||
+ | *# Kill gene, pSB1C3 | ||
+ | *# GFP, pSB1C3 | ||
+ | *# GFP, TetR | ||
+ | * Mini-cultured pET20b and pET26b | ||
+ | |||
+ | ==== Wednesday, 12th ==== | ||
+ | * Observed following from the transformations from 9/11 | ||
+ | *# TetR/GFP-successful with about 250 colonies | ||
+ | *# TetR/Upps 1-successful with about 200 colonies | ||
+ | *# No growth with Kill&pCB1C3, GFP&pCB1C3 | ||
+ | * Prepared colony PCR for TetR&GFP, and TetR&Upps 1 on Amp grid plate (had A-D columns and 1-4 rows) | ||
+ | *# Resuspended single colonies in 4μL deionized water | ||
+ | *# Transferred 3μL colony suspension to Amp grid | ||
+ | *# Stored remaining 1μL as PCR template | ||
+ | *# Grew Amp grid plate in 37C overnight | ||
+ | ''Note 1: GFP/TetR: A and B with 1-4 each'' | ||
+ | |||
+ | ''Note 2: Upps 1/TetR: C and D with 1-4 each'' | ||
+ | * PCR reaction contained following mixture: | ||
+ | ** 1x PCR Rxn | ||
+ | *# 1.0μL of 10x buffer | ||
+ | *# 0.4μL of dNTP | ||
+ | *# 0.4μL of Primer F | ||
+ | *# 0.4μL of Primer R | ||
+ | *# 1.0μL of template mentioned above | ||
+ | *# 0.4μL of tag | ||
+ | *# 6.4μL of deionized water | ||
+ | ** 17x PCR Rxn | ||
+ | *# 17μL of 10x buffer | ||
+ | *# 6.8μL of dNTP | ||
+ | *# 6.8μL of Primer F | ||
+ | *# 6.8μL of Primer R | ||
+ | *# 6.8μL of tag | ||
+ | *# 108.8μL of deionized water | ||
+ | * PCR reactions conducted with following protocol: | ||
+ | *# Denature at 95C for 10 min. | ||
+ | *# Denature at 95C for additional 30 sec. | ||
+ | *# Anneal at 56C for 30 sec. | ||
+ | *# Elongation at 70C for 60 sec. | ||
+ | *# Elongation at 72C for 20 min. | ||
+ | *# Hold at 4C for 39 cycles | ||
+ | * Set up second half composite construction digests for following: | ||
+ | *# Upps 1; Enzyme: EcoRI and SpeI; Buffer E*; Ratio: 1:2; Reaction vol.: 30μL | ||
+ | *# Upps 4; Enzyme: EcoRI and SpeI; Buffer E*; Ratio: 1:2; Reaction vol.: 30μL | ||
+ | *# Upps 5; Enzyme: Xba and Pst; Buffer H; Ratio: 1:1; Reaction vol.: 20μL | ||
+ | *# Upps 6; Enzyme: Xba and Pst; Buffer H; Ratio: 1:1; Reaction vol.: 20μL | ||
+ | *# Kill gene; Enzyme: Xba and Pst; Buffer H; Ratio: 1:1; Reaction vol.: 20μL | ||
+ | *# Linearlized pSB1T3; Enzyme: EcoRI and Pst; Buffer H; Ratio: 1:1; Reaction vol.: 20μL | ||
+ | *# Linearlized pSB1K3; Enzyme: EcoRI and Pst; Buffer H; Ratio: 1:1; Reaction vol.: 20μL | ||
+ | *# Promoterless GFP; Enzyme: Xba; Buffer H; Ratio: N/A; Reaction vol.: 20μL | ||
+ | *# pUC19; Enzyme: EcoRI; Buffer H; Ratio: N/A; Reaction vol.: 20μL | ||
+ | ''Note 3: for * buffers, will use buffer 2 or 4 in future'' | ||
+ | * Reactions took place for 1 hr. in 37C incubation and placed in -20C | ||
+ | * Isolated pGLO and pET20b vectors using standard plasmid isolation protocol | ||
+ | * Prepared glycerol stocks for pET20b and pET26b | ||
+ | ==== Thursday, 13th ==== | ||
+ | * Conducted fphA PCR mutogenesis with following (template = pASKfphA 753bp) | ||
+ | ** 1x PCR Rxn | ||
+ | *# 2.0μL of 10x Buffer | ||
+ | *# 0.4μL of dNTP | ||
+ | *# 0.4μL of Primer F (EcoRI) | ||
+ | *# 0.4μL of Primer R (EcoRI) | ||
+ | *# 1.0μL of template | ||
+ | *# 0.4μL of tag | ||
+ | *# 15.4μL of deionized water | ||
+ | ** 3x PCR Rxn | ||
+ | *# 6.0μL of 10x Buffer | ||
+ | *# 1.2μL of dNTP | ||
+ | *# 1.2μL of Primer F (EcoRI) | ||
+ | *# 1.2μL of Primer R (EcoRI) | ||
+ | *# 1.2μL of tag | ||
+ | *# 46.2μL of deionized water | ||
+ | * Set up 4 min. extension time with 55C for annealing temp for PCR program | ||
+ | * Digested above mixture with Dpn1 at 37C for 1hr. after PCR | ||
+ | * Transformed product into DH5α | ||
+ | * Heat inactivated samples with Spe1 restriction digests from 9/12 at 80C for 20 min. and rest at 65C for 20 min. | ||
+ | * Antartic treated the following (15 min. 37C, 5 min. 65C): | ||
+ | *# Upps 4 | ||
+ | *# Promoterless GFP | ||
+ | *# pUC19 | ||
+ | * Conducted ligations for the following for a total volume of 10μL | ||
+ | *# Upps 4-Kill gene-pSB1K3 | ||
+ | *# Upps 4-Upps 5-pSB1K3 | ||
+ | *# Upps 4-Upps 6-pSB1K3 | ||
+ | *# TetR/Spe (from 9/7)-promoterless GFP | ||
+ | *# pUC19 | ||
+ | * Transformed ligated DNA into DH5α | ||
+ | * Ran gels for PCR Rxn and observed following: | ||
+ | *# A3, B3 had 300 bp (estimated) product | ||
+ | *# B1, B4 had 700 bp (estimated) product | ||
+ | *# Everything else (including marker failed) | ||
+ | *# Can not differentiate between ±TetR | ||
+ | * Minicultured 4 TetR-Upps 1 cultures randomly | ||
+ | ==== Friday, 14th ==== | ||
+ | * Sterilized glycerol for stocks | ||
+ | * Created more LB/Amp plates | ||
+ | * Created more sterile miniculture tubes | ||
+ | * Ran 100 bp optimization gel | ||
+ | ''Note 1: While 2µl DNA visble, should use minimum of 5µl/lane for system (SYBR)'' | ||
+ | * Repeated VF2-VR PCR on TetR&promoterless GFP (denoted A) and TetR&Upps 1 (denoted B) | ||
+ | ** 1x PCR Rxn | ||
+ | **# 1.0μL of 10x Buffer | ||
+ | **# 0.4μL of dNTP | ||
+ | **# 0.4μL of Primer F (EcoRI) | ||
+ | **# 0.4μL of Primer R (EcoRI) | ||
+ | **# 1.0μL of template | ||
+ | **# 0.4μL of tag | ||
+ | **# 6.4μL of deionized water | ||
+ | ** 17x PCR Rxn | ||
+ | **# 17.0μL of 10x Buffer | ||
+ | **# 6.8μL of dNTP | ||
+ | **# 6.8μL of Primer F (EcoRI) | ||
+ | **# 6.8μL of Primer R (EcoRI) | ||
+ | **# 6.8μL of tag | ||
+ | **# 108.8μL of deionized water | ||
+ | ''Note 2: Will pick 7 colonies from each and have 4 minicultures of B-will use 1µl±1 colony'' | ||
+ | * After review of primers, changed PCR program as follows: | ||
+ | *# Increased annealing temp to 60C | ||
+ | *# Increased extension temp to 70C | ||
+ | *# Reduced cycles to 30 | ||
+ | ==== Thursday, 20th ==== | ||
+ | * Reviewed order of custom primer from Invitrogen (primer ordered: 9/17) | ||
+ | * Rehydrated primers | ||
+ | * Ran PCR Rxn with following: | ||
+ | *# Annealing temperature: 61C | ||
+ | *# Elongation time: 3 minutes | ||
+ | *# 30 cycles | ||
+ | |||
+ | ==== Monday, 24th ==== | ||
+ | * Reviewed plates and observed the following: | ||
+ | ** Noted plenty of colonies on both plates, as expected | ||
+ | ** Plates with pSB1C3-J04450 based vector had many pink colonies and 8-10 white clones | ||
+ | ** Considering both vectors were alkaline phosphatase treated, will assume treatment failed for both vectors | ||
+ | ** Will currently disregard Upps 3 based vector transformations | ||
+ | * Picked following colonies for miniculture: | ||
+ | *# pSB1C3-FphA | ||
+ | *# Upps 4-Kill gene | ||
+ | *# Upps 4-Upps 6 | ||
+ | *# TetR-Upps 1 | ||
+ | * Minicultured the following from glycerol stocks | ||
+ | *# Upps 1 | ||
+ | *# Upps 4 | ||
+ | *# TetR | ||
+ | |||
+ | ==== Tuesday, 25th ==== | ||
+ | * Ran a gel to verify following plasmids: | ||
+ | ** Sizing Gel 1 | ||
+ | **# Kill gene 1 (OK) | ||
+ | **# Kill gene 2 (OK) | ||
+ | **# FphA1 (too small - discard) | ||
+ | **# FphA2 (OK) | ||
+ | **# Upps 1 (too big - discard) | ||
+ | **# B0034 RBS (OK, faint) | ||
+ | ** Sizing Gel 2 | ||
+ | **# Upps 4-Kill gene 1 (OK) | ||
+ | **# Upps 4-Kill gene 2 (OK) | ||
+ | **# Upps 4-Upps 6 (OK) | ||
+ | **# Upps 4-Upps 6 (too big - discard) | ||
+ | **# Upps 4 (OK) | ||
+ | **# B0030 RBS.1 (OK, but really faint) | ||
+ | ''Note 1: the parenthesis denote observations/analysis of gel'' | ||
+ | * Isolated plasmids using standard miniprep protocol for the minicultures from previous day | ||
+ | * Set up PCR site directed mutagenesis using pSB1C3-FphA as template | ||
+ | ''Note 2: used protocol from 9/13 and EcoRI primer set'' | ||
+ | * Rehydrated the following using standard iGEM rehydration protocol | ||
+ | *# BBa_J23100 (plate 1, 18C [Amp]) | ||
+ | *# BBa_J23101 (plate 1, 18E [Amp]) | ||
+ | *# Bba_J23102 (plate 1, 18G [Amp]) | ||
+ | * Transformed the above rehydrated biobricks into DH5α | ||
+ | |||
+ | ==== Wednesday, 26th ==== | ||
+ | * Digested the PCR Rxn with DpnI | ||
+ | * Transformed pSB1C3-FphA into DH5α | ||
+ | * Digested the following: | ||
+ | *# BBa_B0034 RBS with SpeI | ||
+ | *# Upps4 with XbaI | ||
+ | *# Kill gene with XbaI | ||
+ | * Ligated the following (was predigested with EcoRI): | ||
+ | *# RBS-Upps 4 | ||
+ | *# RBS-Kill gene | ||
+ | * Gel extracted the bands of the following: | ||
+ | *# RBS-Upp4 Target 2352bp, erroneous 2155bp | ||
+ | *# RBS-Kill Target 3441bp, erroneous 2155bp | ||
+ | * Minicultured the following: | ||
+ | *# Upps 1 | ||
+ | *# Promoterless GFP | ||
+ | *# Promoters 100-102 | ||
+ | |||
+ | ==== Wednesday, 27th ==== | ||
+ | * Made minipreps from minicultures from previous day, ran qualitative gel, and concluded the following: | ||
+ | *# Upps 1 had poor yield | ||
+ | *# Promoterless GFP showed no growth | ||
+ | * Conducted digestions of the following: (yielded 2x volume) | ||
+ | *# RBS with SpeI | ||
+ | *# Promoter 102 with SpeI | ||
+ | *# Upps 1 with XbaI | ||
+ | *# Promoterless GFP with XbaI | ||
+ | * Conducted ligations of the following using digestion with 1x volume of EcoRI: (yielded 2x volume) | ||
+ | *# Promoter 102-Upps 1 | ||
+ | *# Promoter 102-Promoterless GFP | ||
+ | *# RBS-Upps 4 | ||
+ | *# Ribosome Binding Site-Kill gene | ||
+ | * Ran 1x volume on gel to purify and gel extract target band | ||
+ | * Transformed the above in DH5α using standard heat shock protocol | ||
+ | * Observed the PCR site mutagenesis yielded no colonies | ||
+ | |||
+ | ==== Friday, 28th ==== | ||
+ | * Observed that Ribosome Binding Site-Kill gene worked and everything else did not | ||
+ | * Conducted minicultures for the following: | ||
+ | *# Ribosome Bidning Site-Kill gene | ||
+ | *# Promoterless GFP with Kan resistance | ||
+ | *# Upps 1 | ||
+ | *# Upps 4 | ||
+ | * Conducted digestions of the following: | ||
+ | *# Upps 1 with XbaI | ||
+ | *# Inducible Promoter with SpeI | ||
+ | *# Upps 1 with EcoRI/SpeI | ||
+ | *# Upps 4 with XbaI/PstI | ||
+ | *# pSB1A3 (linearized DNA) with EcoRI/PstI | ||
+ | *# pSB3T5 with EcoRI/PstI | ||
+ | * Conducted ligations of the following: | ||
+ | *# Promoter 100-Upps 1 | ||
+ | *# Promoter 101-Upps 1 | ||
+ | *# Promoter 102-Upps 1 | ||
+ | *# Inducible Promoter-Upps 1 | ||
+ | *# Promoter 102-promoterless GFP | ||
+ | *# Upps 1-Upps 4-pSB1A3 | ||
+ | *# Upp1-Upp4-pSB3T5 | ||
+ | * Transformed the above ligated products into DH5α using heat shock protocol | ||
+ | |||
+ | ==== Saturday, 29th ==== | ||
+ | * Observed that Promoter 102-GFP worked and everythign else did not | ||
+ | * Conducted digestions of the following: | ||
+ | *# Upps 1 with EcoRI&SpeI | ||
+ | *# Upps 4-Kill with XbaI&PstI | ||
+ | *# Upps 4-Upps 6 with XbaI&PstI | ||
+ | *# pSB1C3-J04450 with EcoRI&PstI | ||
+ | * Conducted ligations of the following: | ||
+ | *# Upps 1-Upps 4-Kill gene-pSB1C3 destination plasmid | ||
+ | *# Upps 1-Upps 4-Upps6-pSB1C3 destination plasmid | ||
+ | * Transformed the above ligated products into DH5α using a heat shock protocol | ||
+ | * Transformed the following into JM109: | ||
+ | *# Promoter 100-Upps 1 | ||
+ | *# Promoter 101-Upps 1 | ||
+ | *# Promoter 102-Upps 1 | ||
+ | *# Inducible Promoter-Upps 1 | ||
+ | |||
+ | </div> | ||
+ | == October, 2012 == | ||
+ | <div class="content-body-box"> | ||
+ | ==== Monday, 1st ==== | ||
+ | * Observed that all of the plates grew colonies | ||
+ | * Prepared minicultures of the following: | ||
+ | *# Upps 1-Upps 4-Kill gene-pSB1C3 destination plasmid | ||
+ | *# Upps 1-Upps 4-Upps 6-pSB1C3 destination plasmid | ||
+ | *# Promoter 102-Upps 1 | ||
+ | *# Inducible promoter-Upps 1 | ||
+ | |||
+ | ==== Tuesday, 2nd ==== | ||
+ | * Isolated following plasmids by following standard plasmid isolation protocol and submitted parts: | ||
+ | *# pSB1C3-FphA | ||
+ | *# Upps 4-Kill gene | ||
+ | *# Upps 1-Upps 4-Kill gene | ||
+ | *# Upps 1-Upps 4-Upps 6 | ||
+ | *# Promoter 102-Upps 1 | ||
+ | *# Inducible promoter-Upps 1 | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </div> |
Latest revision as of 07:36, 8 January 2013
Columbia Genetics Lab Notebook
July, 2012
Thursday, 5th
- Re-hydrated plasmids with 50µl of LB and Kanamycin solution
- Stored solution at 37°C incubator overnight
Friday, 6th
- Purified pET26b vector using standard DNA purification protocol
Monday, 9th
- Received kill gene Bba-K124017 from plate 3, 20M
- Re-hydrated DNA according to standard iGEM re-hydration protocol
Tuesday, 10th
- Contacted professors at Germany in hopes to receive copies of fungal phytochrome FphA
Wednesday, 11th
- Received confirmation by professors at Germany for FphA to be sent to Columbia University
- Conducted transformation using electroporation with competent bacteria (marked by resistance to Kanamycin)
- Control: 1µl of deionized water with abt. and 60µl of bacteria cells
- Variable: 1µl of re-hydrated kill gene and 60µl of bacteria cells
- Placed both samples after electroporation into 200µl of preprepared LB
- Placed samples in shaker 37°C for 30 minutes
Thursday, 12th
- Grew 1 colony of transformed bacteria in 5mL of Kanamycin and LB solution
- Note: Using pET20b vector over pET26b vector from glycerol stock solution
Friday, 13th
- Isolated 4 samples of plasmid using standard plasmid isolation protocol
- 2 samples: kill gene
- 2 samples: pET20b vector
Monday, 16th
- Re-hydrated two biobrick parts in plasmid pSB2K3 according to standard iGEM re-hydration protocol
- BBa-I16009 (PcyA) from plate 1, 20F
- BBa-I16008 (ho1) from plate 2, 13J
- Electroporated 1µl of each biobrick into separate E. coli at 1800V
- Added 100µl LB broth into each sample
- Placed samples at 33.4°C for 20 minutes
- Samples were plated to be grown overnight
Tuesday, 17th
- Placed 5ml each of LB/Kan into two centrifuge tube for PCB creation
- Label P: PcyA
- Label h: ho1
- Placed samples in 37°C incubator
Wednesday, 18th
- Purified ho1 and PcyA plasmids using standard DNA purification protocol
- Placed purified DNA into glycerol stock (LB/Kan) and stored at -80°C
Thursday, 19th
- Purified GFP using standard DNA purification protocol
- Prepared glycerol stock solution (500µl GFP/500µl 80% glycerol) and stored at -80°C
Tuesday, 24th
- Re-hydrated four biobrick parts according to standard iGEM re-hydration protocol
- Inducible plasmid (pSB1AK3-J04500) from plate 4, 12A
- GFP (pSB1A2-E0040) from plate 1, 14K
- High copy plasmid pSB1T3-J044500 from plate 1, 7A
- Low copy plasmid (pSB3C5-J044500) from plate 1, 3C
- Electroporated competent E. coli with each of the four above genes separetely
- Created Kan, Amp, Cam, Tetra, and Amp/Kan plates
Wednesday, 25th
- Streaked pSB1T3-J04450
- Created LB solution with Kan or Amp or Cam
Thursday, 26th
- Prepared Glycerol stock for inducible promoter, GFP, and low-copy plasmid
- Picked a single colony from pSB1T3-J04450 and let it grow overnight in 37C
- Recorded and measured the DNA concentrations of following at 260nm:
- Kill gene
- PcyA
- ho1
- GFP
- Inducible promoter
- Low copy CAM plasmid
- Followed digestion and ligation protocol; setup explained below:
- Upstream: ho1; Downstream: PcyA; Destination plasmid: Low-copy CAM
- Upstream: Inducible promoter; Downstream: GFP; Destination plasmid: Low-copy CAM
- Upstream: Inducible promoter; Downstream: Kill; Destination plasmid: High-copy TET (pSB1T3)
- After completion, store samples at -20C
Friday, 27th
- Electroporated ligated samples from previous day: GFP, PCB, Kill
- Followed standard plasmid isolation protocol for pSB1T3-J04450 (TET plasmid)
Saturday, 28th
- Check plates from electroporation from previous day
Monday, 30th
- Stored pif3 and phyB that arrived from Sweden
- Relocated and reorganized the iGEM biobrick kit and glycerol stocks
- Picked colonies for following DNA:
- Inducible promotor (pSB1AK3_J04500)
- GFP (pSB1A2_E0040)
- Low copy CAM plasmid (pSB3C5_J04150)
- Added 10µl of ho1 and PcyA to 5ml of antibiotics
- Electroporated following DNA:
- Inducible promoter IPTG/kill gene in BL21 cells
- Inducible promoter IPTG/GFP in BL21 cells
- PCB in α cells
- Transferred successful electroporated cells to eppendorf tube with 100μL of LB and placed in 37C shaker
Tuesday, 31st
- Prepared glycerol stock of the following:
- GFP
- Inducible promoter (IPTG)
- Low copy CAM plasmid
- pcyA
- ho1
- Isolated ho1 and PcyA using standard plasmid isolation protocol
August, 2012
Wednesday, 1st
- Applied IPTG to samples of bacteria with GFP or kill gene and placed back in incubator at 37C
- Electroporated the following plasmids:
- IPTG inducible promoter/kill gene into BL21 cells
- IPTG inducible promoter/GFP into BL21 cells
- phyB into α select cells
- pif3 into α select cells
- Followed the PCR purification protocol for samples 1, 2 listed above
- Chemically transformed the following using standard heat shock protocol provided by Bioline:
- IPTG inducible promoter/kill gene into BL21 cells
- IPTG inducible promoter/GFP into BL21 cells
- Plated PCB onto LB/CAM plate
- Plated Pif3 and phyB on KAN plates
- Plated 500μL of GFP containing cells among two plates each LB/CAM (with and without IPTG)
- Placed all samples in 37C incubator
Thursday, 2nd
- Selected colonies from PhyB from the LB/Kan plate
- Prepared CAM plates
- Selected colonies from GFP control
- Received agar stabs from Uppsala
Friday, 3rd
- Placed 60μL of IPTG to half of control plate for reconfirmation of results
- Streaked GFP (with IPTG inducible promoter) and Pif3 on plates
Saturday, 4th
- Streaked all 6 Uppsala (labelled below) parts from stabs onto plates:
- Upps 1: pSB1K3-B0034-YF1-B0034-FixJ
- Upps 2: pSB1K3-YF1
- Upps 3: pSB1K3-FixJ
- Upps 4: pSB1C3-PfixK2
- Upps 5: pSB1A3-amilCP
- Upps 6: pSB1C3 - amilGFP
- Prepared glycerol stock for phyB (ETHZ)
- Isolated phyB by following standard plasmid isolation protocol
- Electroporated fphA and ho1 from Germany
- Created LB/TET, LB/CAM, LB/Amp, LB/Kan plates
Sunday, 5th
- Pulled colonies from 6 iGEM parts and ho1 from Germany
- Streaked fphA from Germany
- Pulled colonies for Upps 2, 3, 4, 5, 6
- Streaked Upps 1, 3, 4, 6
- Parafilmed all plates and placed in 4C
- Sorted and threw out unnecessary plates
Monday, 6th
- Pulled colonies from fphA and Upps 1
- Isolated plasmids for the following samples following standard plasmid isolation protocol:
- 6 iGEM parts
- Upps 2-6
- ho1
- Reconfirmed that TET plates are valid
Tuesday, 7th
- Prepared glycerol stock and isolated the plasmid using standard plasmid isolation protocol for following:
- fphA from Germany
- Upps 1 (pSB1K3-B0034-YF1-B0034-FixJ)
- Prepared digestion by measuring OD at 260nm for following:
- Upps 1
- Upps 4
- Upps 5
- Upps 6
- Kill Gene
- Inducible promoter containing plasmid
- Prepared digestions and ligated with following setup using the 3A assembly protocol provided from Bioline:
- Upstream: Upps 4; Downstream: Upps 5; Destination Plasmid: High Copy TET plasmid
- Upstream: Upps 4; Downstream: Upps 6; Destination Plasmid: High Copy TET plasmid
- Upstream: Upps 4; Downstream: Kill gene; Destination Plasmid: Low Copy CAM plasmid
- Upstream: Inducible promoter; Downstream: Upps 5; Destination Plasmid: High Copy TET plasmid
- Upstream: Inducible promoter; Downstream: Upps 6; Destination Plasmid: High Copy TET plasmid
- Upstream: Inducible promoter; Downstream: Kill gene; Destination Plasmid: High Copy TET plasmid
Wednesday, 8th
- Pulled colony from GFP streak
- Isolated following using standard plasmid isolation protocol:
- Low Copy CAM plasmid
- High Copy TET plasmid
- Inducible promoter
- Checked optical density and applied necessary dilutions for GFP
- Prepared two samples of GFP: sample with IPTG and sample without IPTG
- Place samples in shaker to grow overnight
Thursday, 9th
- Diluted GFP solutions to match proper optical density
- Pulled colony for ho1 as backup and place in 37C inbucator
Friday, 10th
- Remade 200 mL each of antibiotic solutions for CAM, TET, Kan, Amp
- Conducted digestions and ligations using the 3A assembly method following the protocols provided by bioline of following:
- Upstream: Upps 4; Downstream: Kill gene; Destination Plasmid: High Copy TET plasmid
- Upstream: Inducible promoter; Downstream: Upps 5; Destination Plasmid: Low Copy CAM plasmid
- Upstream: Inducible promoter; Downstream: Upps 6; Destination Plasmid: Low Copy CAM plasmid
- Upstream: Inducible promoter; Downstream; Kill gene; Destination Plamid: Low Copy CAM plasmid
- Followed the butanol purification protocol for ligated material containing inducible promoter from 7th and 10th
Saturday, 11th
- Reviewed the solutions for diluted GFP and observed no significant results
- Reorganized samples in fridges and incubators
Monday, 13th
- Chemically transformed competent cells (BL21) with plasmids below using bioline protocol (used 1/2 of recommended amount)
- IPTG-Upps 5-Low Copy (CAM)
- IPTG-Upps 6-Low Copy (CAM)
- IPTG-Kill gene-Low Copy (CAM)
- CAM control plasmid
- PUC19 control plasmid
- Electroporated competent cells (α-select) with plasmids below using bioline protocol
- Upps 4-Kill gene-High Copy (TET)
- Upps 4-Upps 5-High Copy (TET)
- Upps 4-Upps 6-High Copy (TET)
- PUC19 control plasmid
- High copy TET control plasmid
Note 1: TET control sparked
Note 2: Upps 4-Upps 5-TET sparked
Note 3: Original DNA for Upps 4-Upps 5-TET was pink
- Placed transformed samples in growth media and placed in 37°C shaker
- Made 60ml of 1% agar gel for running gel electrophoresis (2 rows of 12 wells each noted below for gel) to check digestion and ligation
- First row
- 2 log ladder
- Upps 4
- blank
- High Copy TET plasmid
- Upps 4 (2)
- Upps 6
- High Copy TET plasmid (2)
- Upps 4 (3)
- Kill gene
- High Copy TET plasmid (3)
- PCB
- High Copy TET plasmid (4)
- Second row
- 2 log ladder
- Inducible promoter-IPTG
- Kill gene (2)
- High Copy TET plasmid (5)
- Inducible promoter-IPTG (2)
- Upps 6 (2)
- High Copy TET plasmid (6)
- Inducible promoter-IPTG (3)
- Upps 5 (2)
- High Copy TET plasmid (7)
- First row
- Ran gel for 25 minutes at constant 150V
- Took picture under UV light
- Created TET and CAM plates
Tuesday, 14th
Note: Work done below was conducted at the Kanbar Lab at the Cooper Union
- Prepared CAM from .250g of 25mg/mL CAM powder with 10mL of EtOH
- Prepared LB/glucose media
- Heat shocked DH5α competent cells with Inducible promoter/GFP/Low Copy CAM plasmid
Wednesday, 15th
- Diluted bacteria cultures with following plasmids to 200x LB/CAM and placed in 37°C shaker
- IPTG-Upps 5-Low Copy (CAM)
- IPTG-Upps 6-Low Copy (CAM)
- IPTG-Kill gene-Low Copy (CAM)
- Purified above plasmids and CAM control plasmid using standard purification protocol and prepared glycerol stocks
- Added appropriate buffers to IPTG-Upps 5 and IPTG-Upps 6 and centrifuged for 10 minutes to determine for a pellet
- Measured OD 600 of following samples
- IPTG-Upps 5-Low Copy (CAM): .160A
- IPTG-Upps 6-Low Copy (CAM): .038A
- Inserted 1µl of 1M IPTG into cultures
- Placed all samples in 37°C overnight
Note: conducted the below procedures at the Kanbar Lab
- Observed no growth for inducible promoter/GFP/low copy CAM plasmid
Thursday, 16th
- Measured OD 600 for 200x diluted bacterial solution containing plasmids with promoter inducible with IPTG
- IPTG-Upps 5-Low Copy (CAM): .040A
- IPTG-Upps 6-Low Copy (CAM): .032A
- IPTG-Kill gene-Low Copy (CAM): .028A
- Noted that cell concentration was dense, decided to dilute solution with 75µl cells and 925µl LB/CAM solution
- Placed diluted cell solution into 37°C incubator for 40 minutes
- Inserted 1µl of 1M IPTG into cultures
- Placed all samples in 37°C overnight
Friday, 17th
Note: IPTG induced I-U5 and I-U6 appear to give no color change (no expression)
- Electroporate the following genes:
- Upps 4-Upps 6-High copy TET (sparked first time, redid trial)
- Upps 4-Kill-High copy TET
- Upps 4-Kill-Low copy CAM
- pUC19 control DNA (AMP resistance)
- Placed all samples in 37°C overnight
Note2: Only adding 25µl of competent cells instead of 50µl mentioned in the
Saturday, 18th
- Observed none of the electroporated DNA grew, but observed colonies for control
- Placed all samples back in the 37°C incubator
Monday, 20th
- Measured the optical density for the following:
- Upps 6 (with inducible promoter)
- Kill gene
- GFP
- Upps 5 (with inducible promoter)
- Diluted the concentrated primer into a primer stock that would be used for DNA sequencing
- Sent following DNA for sequencing (at Genewiz)
- Inducible promoter-GFP-Low Copy CAM plasmid
- Inducible promoter-Upps 5-Low Copy CAM plasmid
- Inducible promoter-Upps 6-Low Copy CAM plasmid
- Inducible promoter-Kill gene-Low Copy CAM plasmid
- Analyzed gel and concluded following:
- The digestion for TET high copy plasmid did not work
- The digestion for Upps 4, 5, 6 appeared to have worked
- All other digestions are inconclusive results
Tuesday, 21st
- Made additional Amp/CAM and Amp/TET plates
- Measured the optical density for the following:
- Upps 1
- Kill gene
- High copy TET plasmid
- Followed the standard digestion protocol provided by bioline or the following:
- Upps 1 (Upstream)
- Kill gene (Downstream)
- High copy TET plasmid (Destination plasmid)
- Re-organized iGEM boxes by throwing out previous digestions of High Copy TET plasmids
- Re-hydrated following biobricks from standard rehydration protocol:
- Constitutive promoter-BBa_J130002 (P1, 13B)
- Low-copy TET plasmid-PSB3T5 (P1, 7C)
- High-copy CAM plasmid-PSB1C3 (P1, 3A)
- High-copy Amp/CAM plasmid-PSB1AC3 (P1, 9A)
- High-copy Amp/TET plasmid-PSB1AT3 (P1, 13A)
- Made 60ml of 1% agar gel for running gel electrophoresis (2 rows of 12 wells each noted below for gel) to check digestion and ligation
- First row
- 2 log ladder
- old TET high copy plasmid
- old Kill gene
- Upps 5
- IPTG inducible promoter
- Second row
- 2 log ladder
- newly digested TET high copy plasmid
- newly digested Kill gene
- Upps 1
- First row
- Ran gel for 25 minutes at constant 150V
- Took picture under UV light
Wednesday, 22nd
- Conducted ligations for the following using a vector to insert ratio of 1:3
- Sample 1: Inducible promoter (upstream insert)-Kill gene (Downstream insert)-pSB3C5 (vector)
- Sample 2: Inducible promoter (upstream insert)-Upps 5 (Downstream insert)-pSB3C5 (vector)
- Sample 3: Inducible promoter (upstream insert)-Upps 6 (Downstream insert)-pSB3C5 (vector)
- Sample 4: Upps 4 (upstream insert)-Kill gene (Downstream insert)-pSB1T3 (vector)
- Sample 5: Upps 4 (upstream insert)-Upps 5 (Downstream insert)-pSB1T3 (vector)
- Sample 6: Upps 4 (upstream insert)-Upps 6 (Downstream insert)-pSB1T3 (vector)
- Sample 7: Control pSB3C5 (1:0 vector to insert ratio)
- Sample 8: Control pSB1T3 (1:0 vector to insert ratio)
Note: above ligations will be done with T4 buffer and ligase from both the Biobrick kit and Columbia's lab
Note 2: label all sample using iGEM ligase and buffer with prefix N
Note 3: label all sample using Columbia lab's ligase and buffer with B
- Electroporated the following using the standard protocol provided by Bioline:
- Constitutive promoter-BBa_J130002
- Low-copy TET plasmid-PSB3T5
- High-copy CAM plasmid-PSB1C3
- High-copy Amp/CAM plasmid-PSB1AC3
- High-copy Amp/TET plasmid-PSB1AT3
- PUC19 control plasmid
- Chemically transformed the following using the heat shock protocol:
- N1, N2, N3, N7, B1, B2, B3, B7 into BL21 (DE) pLysZ orange cells
- GFP IPTG given by graduate student at Columbia into BL21 (DE) green cells
- PUC19 into BL21 (DE) pLysZ orange cells
- PUC19 into BL21 (DE) green cells
- Purified ligated protocol for electroporation following QIAquick PCR purification protocol
- Plated all chemically transformed and electroporated cells
Thursday, 23rd
Note: Observed no growth with pSB1T3, BL21 pLyse
Note 2: Observed growth with PUC19 and other BL21
- Streaked sample N5
- Found following 3 restriction sites (to be double checked by graduate student):
- NgomIV: CCGCCGGC
- EcoRI: GAATTC
- PstI: CTGCAG
Friday, 24th
- Diluted IPTG driven cultures to 200x with fresh LB/antibiotic solution
- Followed standard plasmid isolation protocol for everything except the following:
- 1 sample of graduate student's GFP
- Sample of pSB1C3 (the high copy CAM plasmid)
- Checked optical densities of samples B1-B3 and graduate student's GFP
- Created two subsamples each of B1-B3; applied 1M IPTG to half of the subsamples
- Applied 1M IPTG to into one of the samples of graduate student's IPTG-GFP
Saturday, 25th
- Checked IPTG "induced" samples and observed no expression
- Measured optical density for following samples and diluted the samples:
- GFP by graduate student-pre-dilution: 2.0, post dilution: 0.710
- IPTG-kill (N1)-pre-dilution: 2.4, post dilution: 0.736
- IPTG-U5 (N2)-pre-dilution: 1.9, post dilution: 0.740
- IPTG-U6 (N5)-pre-dilution: 2.4, post dilution: 0.630
- IPTG-U5 (B2)-pre-dilution: 1.9, post dilution: 0.681
- IPTG-U6 (B3)-pre-dilution: 1.9, post dilution: 0.651
- Applied 1.5μL of 1M IPTG with 1.5mL of each sample and place in 25C shaker
- Followed standard plasmid isolation protocol for N1 and N5
Monday, 27th
- Observe expression of graduate student's GFP
- Measured optical densities for following:
- Ligated samples:
- N1-N3
- B1-B3
- Mini-prepped:
- Upps1-Upps6
- pSB1C3
- pSB1T3
- pSB1AT3
- pSB1AC3
- pSB1A2
- Ligated samples:
- Sent above samples for sequencing
- Followed protocol from bioline for digestion:
- Downstream: Upps 1
- Upstream: Constitutive promoter
- Destination plasmid: pSB3T5
- Destination plasmid: pSB1C3
- Destination plasmid: pSB1AC3
- Destination plasmid: pSB1AT3
- Streaked out Upps 1 from agar stab
Tuesday, 28th
- Reviewed results from the sequencing from Genewiz
- Design and send new primer (nev) for sequencing
- Conducted ligations for the following using a vector to insert ratio of 1:3
- Constitutive promoter (upstream insert)-Upps 1 (downstream insert)-pSB1C3 (vector)
- Constitutive promoter (upstream insert)-Upps 1 (downstream insert)-pSB3T5 (vector)
- Constitutive promoter (upstream insert)-Upps 1 (downstream insert)-pSB1AT3 (vector)
- Constitutive promoter (upstream insert)-Upps 1 (downstream insert)-pSB1AC3 (vector)
- Electroporated the above 4 ligations without purifying the ligations
- Plated cells into respective plates
- Picked colonies for Upps 1-YF1/FixJ
Wednesday, 29th
- Conducted ligations for the following using a vector to insert ratio of 1:3
- Upps 4 (upstream insert)-Upps 5 (downstream insert)-pSB1C3 (vector)
- Upps 4 (upstream insert)-Upps 6 (downstream insert)-pSB1C3 (vector)
- Upps 4 (upstream insert)-Kill gene (downstream insert)-pSB1C3 (vector)
- Upps 4 (upstream insert)-Upps 5 (downstream insert)-pSB1AC3 (vector)
- Upps 4 (upstream insert)-Upps 6 (downstream insert)-pSB1AC3 (vector)
- Upps 4 (upstream insert)-Kill gene (downstream insert)-pSB1AC3 (vector)
- Extracted Upps 1 (Kan) from glycerol stock from -80C freezer
- Followed graduate student's quickchange protocol and labelled N1-N4, E1-E4, P1-P4 with the labeling as following:
- Sample 1: HF only
- Sample 2: HF and 1.5μL DMSO
- Sample 3: GC only
- Sample 4: GC and 1.5μL DMSO
- Prefix N: NgoMIV mutation
- Prefix E: EcoRI mutation
- Prefix P: PsH mutation
Thursday, 30th
- Followed standard plasmid isolation protocol for Upps 1
- Transformed U4-U5, U4-U6, U4-kill in 2 different plasmids
- Ran gels of Quickchange product
- Column 1: miniprepped plasmid
- Column 2-5: N1-N4
- Column 6-9: E1-E4
- Column 10-13: P1-P4
- Re-ligated Constitutive promoter-Upps 1
September, 2012
Wednesday, 5th
Note: All work done from this day on was done at the Kanbar lab at the Cooper Union
- Single digested the J13002
- Placed the digest above and other digests to be run on gels into -20C freezer
- Made 4 50mL 1% agarose gels
- Followed standard plasmid isolation protocol for kill gene
Friday, 7th
- Ran two gels to check digests
- Gel Number 1
- Marker
- Upps 1
- Inducible promoter 1
- Inducible promoter 2
- pSB3T5
- pSB1C3
- Low Copy Vector 1
- Low Copy Vector 2
- Gel Number 2
- Marker
- Kill gene
- PcyA
- TetR 1
- TetR 2
- GFP from biobrick kit
- GFP from Tushar
- GFP from Kevin
- Gel Number 1
- Conducted digestions for the following:
- Inducible promoter digested with EcoRI&Spe with Buffer E and Buffer Multi (for 30 min.)
- TetR digested with EcoRI with Buffer H
- TetR digested with Spe with Buffer B
- Upps 1 digested with Xba&Pst with Buffer H
- Kill gene digested with Xba&Pst with Buffer H
- GFP digested with Xba&Pst with Buffer H
- pSB3T5 with EcoRI&Pst with Buffer H
- Low copy plasmid 2 digested with EcoRI&Pst with Buffer H
- pSB1C3 digested with EcoR&Pst with Buffer H
Note: Digests 7-9 was done in Antartic Phosphotase Treat 30 min in 37C and 15 min in 65C
- Conducted ligations for the following (followed by chemical transformations:
- Upstream: Inducible promoter; Downstream: Upps 1; Destination plasmid: Low copy plasmid 2
- Upstream: Inducible promoter; Downstream: Kill; Destination plasmid: Low copy plasmid 2
- Upstream: Inducible promoter; Downstream: GFP; Destination plasmid: Low copy plasmid 2
- Conducted additional chemical transformations with the following vectors:
- pET20b
- pET26b
- pUC19
- Conducted following additional ligations, reference numbers correlate to numbers shown above for this day's digestions
- Label A: 2 and 9
- Label B: 3 and 4 and 9
- Label C: 1 and 4 and 8
- Label D: 1 and 5 and 8
- Label E: 1 and 6 and 8
- Label F: 3 and 4
- Label G: 3 and 5
- Label H: 3 and 6
Tuesday, 11th
- Observed the following:
- Inducible promoter-GFP-Low Copy CAM Plasmid had 3 pink clones (IPTG applied)
- Inducible promoter-GFP-Low Copy CAM Plasmid had 3 pink clones (NO IPTG)
- Inducible promoter-Upps 1-Low Copy CAM Plasmid had 32 pink clones
- Inducible promoter-Kill gene-Low Copy CAM Plasmid had 50 pink clones
Note 1: Destination plasmid/vector contained J04450 biobrick part, explained pink clones
Note 2: All vectors were all treated with Antartic Phophotase, expect no religation of J04450 with LC vector
- Digested Kill gene and GFP with E and P
- AP Treat Kill gene with X and P, GFP with X and P
- Conducted ligations for the following:
- GFP (E and P) with pSB1C3 (E and P, AP treated)
- GFP (X and D, AP treated) with TetR/s
- Condcuted transformations for the following:
- Ligation F from 9/7, TetR, Upps 1(X and P)
- Kill gene, pSB1C3
- GFP, pSB1C3
- GFP, TetR
- Mini-cultured pET20b and pET26b
Wednesday, 12th
- Observed following from the transformations from 9/11
- TetR/GFP-successful with about 250 colonies
- TetR/Upps 1-successful with about 200 colonies
- No growth with Kill&pCB1C3, GFP&pCB1C3
- Prepared colony PCR for TetR&GFP, and TetR&Upps 1 on Amp grid plate (had A-D columns and 1-4 rows)
- Resuspended single colonies in 4μL deionized water
- Transferred 3μL colony suspension to Amp grid
- Stored remaining 1μL as PCR template
- Grew Amp grid plate in 37C overnight
Note 1: GFP/TetR: A and B with 1-4 each
Note 2: Upps 1/TetR: C and D with 1-4 each
- PCR reaction contained following mixture:
- 1x PCR Rxn
- 1.0μL of 10x buffer
- 0.4μL of dNTP
- 0.4μL of Primer F
- 0.4μL of Primer R
- 1.0μL of template mentioned above
- 0.4μL of tag
- 6.4μL of deionized water
- 17x PCR Rxn
- 17μL of 10x buffer
- 6.8μL of dNTP
- 6.8μL of Primer F
- 6.8μL of Primer R
- 6.8μL of tag
- 108.8μL of deionized water
- PCR reactions conducted with following protocol:
- Denature at 95C for 10 min.
- Denature at 95C for additional 30 sec.
- Anneal at 56C for 30 sec.
- Elongation at 70C for 60 sec.
- Elongation at 72C for 20 min.
- Hold at 4C for 39 cycles
- Set up second half composite construction digests for following:
- Upps 1; Enzyme: EcoRI and SpeI; Buffer E*; Ratio: 1:2; Reaction vol.: 30μL
- Upps 4; Enzyme: EcoRI and SpeI; Buffer E*; Ratio: 1:2; Reaction vol.: 30μL
- Upps 5; Enzyme: Xba and Pst; Buffer H; Ratio: 1:1; Reaction vol.: 20μL
- Upps 6; Enzyme: Xba and Pst; Buffer H; Ratio: 1:1; Reaction vol.: 20μL
- Kill gene; Enzyme: Xba and Pst; Buffer H; Ratio: 1:1; Reaction vol.: 20μL
- Linearlized pSB1T3; Enzyme: EcoRI and Pst; Buffer H; Ratio: 1:1; Reaction vol.: 20μL
- Linearlized pSB1K3; Enzyme: EcoRI and Pst; Buffer H; Ratio: 1:1; Reaction vol.: 20μL
- Promoterless GFP; Enzyme: Xba; Buffer H; Ratio: N/A; Reaction vol.: 20μL
- pUC19; Enzyme: EcoRI; Buffer H; Ratio: N/A; Reaction vol.: 20μL
Note 3: for * buffers, will use buffer 2 or 4 in future
- Reactions took place for 1 hr. in 37C incubation and placed in -20C
- Isolated pGLO and pET20b vectors using standard plasmid isolation protocol
- Prepared glycerol stocks for pET20b and pET26b
Thursday, 13th
- Conducted fphA PCR mutogenesis with following (template = pASKfphA 753bp)
- 1x PCR Rxn
- 2.0μL of 10x Buffer
- 0.4μL of dNTP
- 0.4μL of Primer F (EcoRI)
- 0.4μL of Primer R (EcoRI)
- 1.0μL of template
- 0.4μL of tag
- 15.4μL of deionized water
- 3x PCR Rxn
- 6.0μL of 10x Buffer
- 1.2μL of dNTP
- 1.2μL of Primer F (EcoRI)
- 1.2μL of Primer R (EcoRI)
- 1.2μL of tag
- 46.2μL of deionized water
- Set up 4 min. extension time with 55C for annealing temp for PCR program
- Digested above mixture with Dpn1 at 37C for 1hr. after PCR
- Transformed product into DH5α
- Heat inactivated samples with Spe1 restriction digests from 9/12 at 80C for 20 min. and rest at 65C for 20 min.
- Antartic treated the following (15 min. 37C, 5 min. 65C):
- Upps 4
- Promoterless GFP
- pUC19
- Conducted ligations for the following for a total volume of 10μL
- Upps 4-Kill gene-pSB1K3
- Upps 4-Upps 5-pSB1K3
- Upps 4-Upps 6-pSB1K3
- TetR/Spe (from 9/7)-promoterless GFP
- pUC19
- Transformed ligated DNA into DH5α
- Ran gels for PCR Rxn and observed following:
- A3, B3 had 300 bp (estimated) product
- B1, B4 had 700 bp (estimated) product
- Everything else (including marker failed)
- Can not differentiate between ±TetR
- Minicultured 4 TetR-Upps 1 cultures randomly
Friday, 14th
- Sterilized glycerol for stocks
- Created more LB/Amp plates
- Created more sterile miniculture tubes
- Ran 100 bp optimization gel
Note 1: While 2µl DNA visble, should use minimum of 5µl/lane for system (SYBR)
- Repeated VF2-VR PCR on TetR&promoterless GFP (denoted A) and TetR&Upps 1 (denoted B)
- 1x PCR Rxn
- 1.0μL of 10x Buffer
- 0.4μL of dNTP
- 0.4μL of Primer F (EcoRI)
- 0.4μL of Primer R (EcoRI)
- 1.0μL of template
- 0.4μL of tag
- 6.4μL of deionized water
- 17x PCR Rxn
- 17.0μL of 10x Buffer
- 6.8μL of dNTP
- 6.8μL of Primer F (EcoRI)
- 6.8μL of Primer R (EcoRI)
- 6.8μL of tag
- 108.8μL of deionized water
- 1x PCR Rxn
Note 2: Will pick 7 colonies from each and have 4 minicultures of B-will use 1µl±1 colony
- After review of primers, changed PCR program as follows:
- Increased annealing temp to 60C
- Increased extension temp to 70C
- Reduced cycles to 30
Thursday, 20th
- Reviewed order of custom primer from Invitrogen (primer ordered: 9/17)
- Rehydrated primers
- Ran PCR Rxn with following:
- Annealing temperature: 61C
- Elongation time: 3 minutes
- 30 cycles
Monday, 24th
- Reviewed plates and observed the following:
- Noted plenty of colonies on both plates, as expected
- Plates with pSB1C3-J04450 based vector had many pink colonies and 8-10 white clones
- Considering both vectors were alkaline phosphatase treated, will assume treatment failed for both vectors
- Will currently disregard Upps 3 based vector transformations
- Picked following colonies for miniculture:
- pSB1C3-FphA
- Upps 4-Kill gene
- Upps 4-Upps 6
- TetR-Upps 1
- Minicultured the following from glycerol stocks
- Upps 1
- Upps 4
- TetR
Tuesday, 25th
- Ran a gel to verify following plasmids:
- Sizing Gel 1
- Kill gene 1 (OK)
- Kill gene 2 (OK)
- FphA1 (too small - discard)
- FphA2 (OK)
- Upps 1 (too big - discard)
- B0034 RBS (OK, faint)
- Sizing Gel 2
- Upps 4-Kill gene 1 (OK)
- Upps 4-Kill gene 2 (OK)
- Upps 4-Upps 6 (OK)
- Upps 4-Upps 6 (too big - discard)
- Upps 4 (OK)
- B0030 RBS.1 (OK, but really faint)
- Sizing Gel 1
Note 1: the parenthesis denote observations/analysis of gel
- Isolated plasmids using standard miniprep protocol for the minicultures from previous day
- Set up PCR site directed mutagenesis using pSB1C3-FphA as template
Note 2: used protocol from 9/13 and EcoRI primer set
- Rehydrated the following using standard iGEM rehydration protocol
- BBa_J23100 (plate 1, 18C [Amp])
- BBa_J23101 (plate 1, 18E [Amp])
- Bba_J23102 (plate 1, 18G [Amp])
- Transformed the above rehydrated biobricks into DH5α
Wednesday, 26th
- Digested the PCR Rxn with DpnI
- Transformed pSB1C3-FphA into DH5α
- Digested the following:
- BBa_B0034 RBS with SpeI
- Upps4 with XbaI
- Kill gene with XbaI
- Ligated the following (was predigested with EcoRI):
- RBS-Upps 4
- RBS-Kill gene
- Gel extracted the bands of the following:
- RBS-Upp4 Target 2352bp, erroneous 2155bp
- RBS-Kill Target 3441bp, erroneous 2155bp
- Minicultured the following:
- Upps 1
- Promoterless GFP
- Promoters 100-102
Wednesday, 27th
- Made minipreps from minicultures from previous day, ran qualitative gel, and concluded the following:
- Upps 1 had poor yield
- Promoterless GFP showed no growth
- Conducted digestions of the following: (yielded 2x volume)
- RBS with SpeI
- Promoter 102 with SpeI
- Upps 1 with XbaI
- Promoterless GFP with XbaI
- Conducted ligations of the following using digestion with 1x volume of EcoRI: (yielded 2x volume)
- Promoter 102-Upps 1
- Promoter 102-Promoterless GFP
- RBS-Upps 4
- Ribosome Binding Site-Kill gene
- Ran 1x volume on gel to purify and gel extract target band
- Transformed the above in DH5α using standard heat shock protocol
- Observed the PCR site mutagenesis yielded no colonies
Friday, 28th
- Observed that Ribosome Binding Site-Kill gene worked and everything else did not
- Conducted minicultures for the following:
- Ribosome Bidning Site-Kill gene
- Promoterless GFP with Kan resistance
- Upps 1
- Upps 4
- Conducted digestions of the following:
- Upps 1 with XbaI
- Inducible Promoter with SpeI
- Upps 1 with EcoRI/SpeI
- Upps 4 with XbaI/PstI
- pSB1A3 (linearized DNA) with EcoRI/PstI
- pSB3T5 with EcoRI/PstI
- Conducted ligations of the following:
- Promoter 100-Upps 1
- Promoter 101-Upps 1
- Promoter 102-Upps 1
- Inducible Promoter-Upps 1
- Promoter 102-promoterless GFP
- Upps 1-Upps 4-pSB1A3
- Upp1-Upp4-pSB3T5
- Transformed the above ligated products into DH5α using heat shock protocol
Saturday, 29th
- Observed that Promoter 102-GFP worked and everythign else did not
- Conducted digestions of the following:
- Upps 1 with EcoRI&SpeI
- Upps 4-Kill with XbaI&PstI
- Upps 4-Upps 6 with XbaI&PstI
- pSB1C3-J04450 with EcoRI&PstI
- Conducted ligations of the following:
- Upps 1-Upps 4-Kill gene-pSB1C3 destination plasmid
- Upps 1-Upps 4-Upps6-pSB1C3 destination plasmid
- Transformed the above ligated products into DH5α using a heat shock protocol
- Transformed the following into JM109:
- Promoter 100-Upps 1
- Promoter 101-Upps 1
- Promoter 102-Upps 1
- Inducible Promoter-Upps 1
October, 2012
Monday, 1st
- Observed that all of the plates grew colonies
- Prepared minicultures of the following:
- Upps 1-Upps 4-Kill gene-pSB1C3 destination plasmid
- Upps 1-Upps 4-Upps 6-pSB1C3 destination plasmid
- Promoter 102-Upps 1
- Inducible promoter-Upps 1
Tuesday, 2nd
- Isolated following plasmids by following standard plasmid isolation protocol and submitted parts:
- pSB1C3-FphA
- Upps 4-Kill gene
- Upps 1-Upps 4-Kill gene
- Upps 1-Upps 4-Upps 6
- Promoter 102-Upps 1
- Inducible promoter-Upps 1