Team:Nevada/Notebook

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Team Nevada Notebook

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Contents 1 APRIL 2 MAY 3 JUNE 4 JULY 5 AUGUST 6 SEPTEMBER 7 OCTOBER

APRIL

Week of April 11-17:

• Christian
  • Transformed pBIB into Top 10 Cells

• Bryson, Michael, Senny, Tyler
  • Made tobacco cell (NT-1) media in Dr. Shintani's lab

Week of April 18-24:

• Bryson, Christian
  • EcoRI digest of pBIB
  • Made Na acetate buffer
• Christian
  • Ran gel of EcoR1 Digested pBIB

MAY

Week of April 25-May 1:

• Bryson
  • Ran agarose gel of EcoRI digest

• Matthew
  • Team collaborated on pBIB assignment. Too many chefs in the kitchen.
  • We decide to each tackle the pBIB problem in parallel

Week of May 2-8:

• Elaine
  • Ran 0.8% agarose gel of EcoRI digest
  • Made LB/KAN plates
  • Spread for colonies
  • Did miniprep for pBIB liquid cultures

• Matthew
  • Miniprepped pBIB
  • Ran EcoRI digest and Klenow
  • Ethanol precipitated and ligated

Week of May 9-15:

• Christian
  • EcoR1/Xba1 Digest of pBIB
  • Did Mini prep on 4 cultures

• Bryson
  • Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.
  • Did minipreps on additional pBIB liquid cultures.

• Elaine
  • Did XbaI and EcoRI digest of pBIB
  • Ran 2 0.8% gels of each digest
  • Did a miniprep and a Phenol:chloroform clean-up
  • Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB

• Matthew
  • Ligated and transformed
  • Screened colonies, miniprepped, and digested, ran on gel
  • No candidates had EcoRI eliminated

Week of May 16-22:

• Christian
  • Generated glycerol stock of pBIB transformed Top 10 cells
  • Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates
  • Ran samples on gel after digest with EcoR1

• Bryson
  • Klenow reactions of EcoRI digests
  • Phenol:chloroform cleanup of pBIB prior to ligation
  • Blunt-end ligation of klenowed pBIB

• Randy Pares and Vidim Gladwell
  • Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.

• Elaine
  • Made LB/KAN plates
  • Made 50mg/ml stock of KAN
  • Made 1X TAE buffer

• Matthew
  • Digested and tested more colonies, none worked
  • Started over with pBIB and Klenow
  • Ethanol precipitated and ligated
  • Transformed
  • Screened colonies, miniprepped, and digested, ran on gel
  • No candidates had EcoRI eliminated

Week of May 23-29:

• Christian
  • Miniprep on pBIB transformed Top 10 cells using "low copy #" modification
  • Digested with EcoR1 and HindIII

• Bryson
  • Incubated 50 mL liquid culture of E. coli with pBIB (samples 3).
  • Miniprepped sample 3, using modified protocol for large/low-copy plasmids.
  • EcoRI digest of uncut sample 3
  • Prepared 5 mM dNTP stock

• Elaine
  • Incubated 50 mL liquid culture of E. coli with pBIB (sample 4 & sample 5)
  • Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids
  • Nanodrop of DNA recovery of miniprepped sample 4 & sample 5

• Randy and Vadim
  • Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22
  • Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene
  • Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit

• Matthew
  • Investigated alternative ways of eliminating EcoRI site.
  • Want to try hexameric linkers

JUNE

Week of May 30-June 5:

• Christian
  • Miniprep Top 10 Cells transformed with pBIB

• Bryson
  • HinD3 digest of uncut sample 3
  • Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI
  • Pooled uncut samples 3, 4 and 5 (pBIB-pool)
  • Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)
  • EcoRI digest of pBIB-pool and pBIB-maxi
  • Ran 0.8% agarose gel of EcoRI digests
  • Klenow reactions of pBIB-pool and pBIB-maxi
  • Made glycerol stocks of pBIB samples 1-5

• Elaine
  • EcoRI digest of uncut pBIB sample 4 and 5
  • HinD3 digest of uncut sample 4 & sample 5 as a positive control
  • Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful
  • Ethanol precipitation of the EcoRI digests of sample 4 & sample 5
  • Nanodrop resulted to a low DNA content
  • Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep
  • Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest
  • Modified all protocols of the Binary vector

• Randy and Vadim
  • Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)
  • Programmed thermal cycler for PCR of ccdB gene
  • Ran PCR for ccdB
  • Prepared LB/KAN Broth
  • Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel
    • Reaction was unsuccessful

• Matthew
  • Miniprepped pBIB
  • Ran EcoRI digests
  • Ran ethanol precipitation
  • Ligated with several concentrations of hexameric linkers designed to destroy site
  • Ran transformation, no colonies
    • Believed procedures/conditions ran on ligation and transformation not ideal

Week of June 6-12:

• Christian
  • Klenow reaction on EcoR1 digested pBIB
  • T4 ligation on Klenow products

• Bryson
  • Ligation reactions for pBIB-pool and pBIB-maxi
  • Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB
  • Transformed Top-10 Cells with modified pBIB (designated pBIB#)
    • Obtained two colonies after overnight incubation
  • Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate

• Randy and Vadim
  • Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI
  • Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB
  • Modified thermal cycler conditions for PCR of ccdB gene
  • Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)
  • Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene

• • Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit

• Matthew
  • Miniprepped pBIB
  • Ran Digestions and ethanol precipitation
  • Ligated with hexameric linkers
  • Transformed, had some colonies

Week of June 13-19:

• Christian
  • EcoR1 digest of ligation product
  • Transformed Top 10 Cells with pBIB from ligation and plated
  • Inoculated 30 colonies
  • UNR mini prep on all 30 colonies
  • Digested Samples with HindIII and EcoR1
  • QIAGEN mini prep on samples 11,12 &13
  • Digested Samples with HindIII and EcoR1

• Bryson
  • Prepared liquid cultures of pBIB# 1 and pBIB# 2
  • Miniprepped liquid cultures of pBIB#
  • EcoRI and HinDIII digests of pBIB#
  • Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#
  • Single-colony streaked pBIB# 2 on a fresh LB-Kan plate

• Elaine
  • Worked with Chris to incubate 30 liquid cell cultures
  • Ran 0.8% gels of all 30 samples

• Randy and Vadim
  • Topocloned ccdB gene into TOPO PCR Blunt II vector
  • Determined concentration of pBIB maxipreps using PicoGreen analysis
  • Single-colony isolated nine colonies of TOPO-cloned ccdB gene
  • Miniprepped cultures of ccdB gene in TOPO-clone
  • Digested minipreps using EcoRI
  • Ran 1% gel for digested minipreps
  • Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9
  • Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector

• Matthew
  • Miniprepped candidates and digested them
  • Ran on gel, some had strange bands, I want to retest
  • Colonies appear to not have eliminated EcoRI

Week of June 20-26:

• Christian
  • Ran gel of Sample 11

• Bryson
  • Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate
  • Started 20 mL liquid cultures of 2-1, 2-2, and 2-3
  • Miniprepped samples
  • EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony
  • 0.8% agarose gel of digests

• Randy and Vadim
  • Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit
  • Nanodropped minipreps
  • Digested minipreps using EcoRI
  • Ran 1% gel for digested minipreps
  • Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added
  • Analyzed ccdB samples using Vector NTI (Invitrogen)
  • Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added

• Elaine
  • Primer Design of RD29A

• Matthew
  • Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI
  • Team reevaluated its standing, given a month of failed attempts to modify pBIB
  • We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak
  • I design primers to extract parts from iGEM vectors with Kozak sequences

JULY

Week of June 27-July 3:

• Randy and Vadim
  • Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit
  • Prepared thermal cycler protocol for pBIB vector
  • Performed multiple PCR on pBIB vector
  • Transformed ccdB into NEB cells unsuccessfully
  • Made chlorophenocol resistant agar plates
  • Agarose gel analyzed PCR products

• Matthew
  • Transformed colonies with iGEM parts, miniprepped.
  • Attempted PCR on EYFP, Failed. Reevaluated protocol.

Week of July 4-10:

• Randy and Vadim
  • Agarose gel analyzed PCR products
  • Transformed ccdB into NEB, OmniMax, and DEB cells
  • Made Kanamycin resistant LB plates

• Elaine
  • Transformed ccdB into NEB, OmniMax, and DEB cells
  • Made LB/KAN plates
  • Took picture of ccdB Sensitivity Experiment 1 Results
  • GFP Primer Design

Week of July 11-17:

• Elaine
  • Made LB/Amp plates

• Randy and Vadim
  • TOPOcloned pBIB fragment
  • Attempted to ligate ccdB into iGEM vector pSB1C3
  • Single colony streaked ccdB transformation
  • Made Chloramphenicol broth
  • Performed PCR on pBIB cell lines
  • Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI
  • Miniprepped ccdB colonies
  • Gel analyzed pBIB PCR product
  • Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates
  • Made 50x TAE buffer
  • Miniprepped pSB1C3+RFP vector

• Matthew
  • Processed Miniprepped iGEM colonies for mCherry and EYFP
  • Set up PCR for mCherry, EYFP, and NOS

Week of July 18-24:

• Elaine
  • PCR of GFP
  • Ran agarose gel analysis of GFP PCR product

• Randy and Vadim
  • Amplified pBIB fragment using PCR
  • Gel analyzed pBIB PCR product
  • Attempted ligation of ccdB into iGEM vector pSB1C3
  • Transformed ligation into NEB cells
  • Selected supposed colonies with ccdB+pSB1C3

• Matthew
  • Analyzed PCR fragments on gels for mCherry, EYFP, and NOS

 

 

 

PCR results: worked for eyfp and mcherry with bands around 700bp.

From Left: Lane 1 - Lamda Hind III Mlc. Marker.

Lane 2 - Uncut iGem vector

Lane 3 - EYFP

Lane 4 - EYFP

Lane 5 - EYFP

Lane 6 - mCherry

Lane 7 - mCherry

Lane 8 - mCherry

Lane 9 - 100bp ladder

 

 

 

 

• • NOS PCR did not appear to work.
  • Performed transformation protocol with TOPO vector
  • Performed screening on Kan and miniprepped

Week of July 25-31:

• Christian
  • Transformed Top 10 cells with pBIB and pMA (containing rd29A)

• Elaine
  • GFP Transformation
  • Cell Culture of GFP
  • Miniprep of GFP
  • EcoR 1 & Pst 1 Digest of GFP
  • Ran 1.2% agarose gel analysis of the GFP digest

• The "GFP E0040" gel analysis was successful for the expected bands of GFP showed at ~720 bp when digested with EcoR 1 & Pst 1 (labeled Cut #1, Cut #2, Cut#3, Cut#4).
  • PCR of GFP

• Randy and Vadim
  • Miniprepped ccdB+pSB1C3 colonies
  • Digested minipreps with EcoRI and PstI
  • Performed colony PCR on pSB1C3 colonies

• Matthew
  • Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO
  • 1 possible candidate each for mChery, EYFP, and NOS.
    • Nos bands did not show but sent one for sequencing anyway

AUGUST

Week of August 1-7:

• Christian
  • Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region
  • Mini prep on pBIB and pMA inoculations

• Elaine
  • Ran 1.2% Agarose Gel analysis of GFP PCR product

• The "GFP PCR" gel analysis was successful for the expected bands of GFP + Primers showed at ~ 804bp for all PCR products (labeled 1 ng, 5ng, 10ng, & ~826ng)
  • Topocloned GFP PCR product
  • Cell Culture of GFP Topocloned colonies
  • Streaked colony plate
  • Miniprep of GFP Topocloned colonies
  • EcoR I digestion of GFP Topocloned
  • Ligation Test: GFP original vector digested with EcoR I

• Samantha and Christian
  • pMA and pBIB liquid cultures and mini preps

• Bryson
  • Designed and ordered primers for the 35S promoter
  • Made spectymycin plates
  • Transformed DB3.1 cells with pK7FWG2
  • Liquid cultures and minipreps to isolate pK7FWG2

• Randy and Vadim
  • Grew ccdB 8-2 on Kan. resistant plates
  • Gel analyzed colony PCR product from July 29
  • Made additional LB broth
  • Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst
    • Ligation was unsuccessful
  • Performed PCR cleanup on ccdB

Week of August 8-14:

• Elaine
  • Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest

• The "GFP in Topo Vector" gel analysis was successful for the EcoR1 digested (cut samples labeled C1, C2, C3, C4, C5, & C6) samples shows bands at ~804bp (GFP + primers) and the Topo bands at ~ 3,519bp.
  • Ligation Test: Ligated EcoR I digest GFP original vector
  • Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells
  • Cell culture and streaked colonies of RFP in PSB1C3 vector
  • Miniprepped RFP in PSB1C3 vector
  • Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI
  • Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors
  • Ligated the RFP & GFP in either PSB1C3 & Topo Vectors
  • Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates

• Samantha and Christian
  • Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI
  • Ligation of triple digest products

• Bryson
  • PCR of pK7FWG2

• Randy and Vadim
  • Made and aliquoted ligase buffer, ATP, and DTT for use for ligation
  • Ligated ccdB+pSB1C3, did not use EtOH precipitation
  • Miniprepped ccdB+pSB1C3 ligation
  • Digested and gel analyzed ligation using EcoRI and Pst
    • Ligation was unsuccessful
  • Ligated ccdB+pSB1C3 using EtOH precipitation

Week of August 15-21:

• Elaine
  • Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates
  • Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors
  • Digested GFP in PSB1C3 vectors with EcoR1 & PstI
  • Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI

• The "GFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed at colony #17 (Digested/Cut sample labeled C17) the GFP (720) + primers [which includes the KpnI (GGT ACC = 6bp), Kozak sequence (AAA AAA AAA ACA = 12bp), & Translationsal Stop codon (TAATAA = 6bp)] band is at ~804bp and the pSB1C3 bands at ~2,072bp.

• Bryson
  • 1.2% gel to confirm presence of amplicon
  • Topocloning of amplicon and transformation of Topo vector into DB3.1 cells

• Randy and Vadim
  • Miniprepped ligation from Aug. 13
  • Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst
    • Ligation was unsuccessful
  • Ligated ccdB+pSB1C3 using EtOH precipitation
  • Made Chlor. resistant plates
  • Autoclaved labware

Week of August 22-28:

• Christian
  • Ran PCR on genomic Arabidopsis thaliana DNA using custom primers
  • Ran gel of PCR product, no bands corresponding to DREB1C

• Elaine
  • Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)
  • Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony #17
  • Miniprepped 8 cell cultures and nanodrop
  • Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)

• Bryson
  • Isolated and streaked 6 colonies
  • Liquid cultures and minipreps of samples
  • Digest of samples with EcoRI and PstI to confirm presence of insert

• Randy and Vadim
  • Miniprepped colonies from ligation from Aug. 20
  • Digested and gel-analyzed ccdB+pSB1C3 ligation
    • Ligation was unsuccessful
  • Ligated ccdB+pSB1C3 using EtOH precipitation
  • Made Chlor. resistant plates

• Richard and Nick
  • Designed BioBrick-compatible primers to amplify CD2+-Promoter AtMRP3 from A. thaliana genomic DNA

SEPTEMBER

Week of August 29-September 4:

• Elaine
  • Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)
  • Analysis of sequence of GFP (PSB1C3)
  • Sequencing analysis of colony #11 & #17

• Bryson
  • Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation
  • Miniprepped cultures and eluted in 300 microliters
  • 50 microliter digests done
    • 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids
    • Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction

• Randy and Vadim
  • Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27
    • Ligation was unsuccessful
  • Performed PCR on ccdB
  • Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation

• Matthew
  • Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.
  • Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo
  • Screened colonies on Kan and Amp, selected several to miniprep
  • Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP

Week of September 5-11:

• Elaine
  • Sequenced 4 tubes of #17 of GFP in PSB1C3
  • Digested rd29A with Hind III & MfeI-HF (for Chris' Project)

• Bryson
  • Transformation of ligation reaction
  • Plated on chloramphenicol
  • Selected four white colonies and single-colony streaked them
  • 4 mL liquid cultures of colonies
  • Miniprepped samples and eluted in 50 microliters
  • 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3

• Randy and Vadim
  • Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells
    • Transformation unsuccessful
  • Made Chlor. resistant plates
  • Made new 3M NaOH stock

• Matthew
  • Miniprepped candidates for mCherry and EYFP in Topo vector
  • Ran digests and gels on topo candidates and submitted them for sequencing
  • Sequencing Results came back positive for mCherry and EYFP in Topo
    • mCherry showed a point mutation but it should not have an effect on translation

• Richard and Nick
  • Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family
  • Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter
  • Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers

Week of September 12-18:

• Christian
  • PCR of DREB1C with custom primers using extaq instead of platinum taq using Arabidopsis thaliana columbian
  • Ran gel of PCR product

• Bands showed at ~500 bp for all samples
  • Sample 2 selected for Topo cloning

• Elaine
  • rd29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest
  • Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3)
  • Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3)
  • Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)

• Randy and Vadim
  • Miniprepped ccdB with Mfe site in TOPO vector
  • Miniprepped RFP in pSB1C3
  • Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst
  • Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst
  • Gel purified ccdB and pSB1C3 fragments from digested samples

• Samantha
  • Gel extraction of RD29A

• Matthew
  • Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak
  • Several candidates possible for sequencing for EYFP and 3 candidates for mCherry

 

 

EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb

From Left: Lane 1 - 100 bp ladder. Did not show

Lane 2 - Uncut EYFP candidate 1

Lane 3 - EYFP candidate 1

Lane 4 - Uncut EYFP candidate 2

Lane 5 - EYFP candidate 2

Lane 6 - Uncut EYFP candidate 3

Lane 7 - EYFP candidate 3

Lane 8 - Uncut EYFP candidate 4

Lane 9 - EYFP candidate 4

 

 

 

 

 

 

 

mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb

From Left: Lane 1 - 100 bp ladder. Did not show

Lane 2 - Uncut mCherry candidate 1

Lane 3 - mCherry candidate 1

Lane 4 - Uncut mCherry candidate 2

Lane 5 - mCherry candidate 2

Lane 6 - Uncut mCherry candidate 3

Lane 7 - mCherry candidate 3

Lane 8 - Uncut mCherry candidate 4

Lane 9 - mCherry candidate 4

 

 

 

 

Week of September 19-25:

• Elaine
  • 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry
  • 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.
  • Streaked single colonies [rd29A + RFP (PSB1C3)] and did Cell cultures
  • Miniprepped [rd29A + RFP (PSB1C3)]and digested with EcoR I and Pst I
  • Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel

• The "rd29A promoter + RFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed the rd29A + RFP bands at ~1,529 bp and the pSB1C3 bands at ~2,072bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C1-2, C1-7, C1-9).
  • Submitted [rd29A + RFP (PSB1C3)] for sequencing

• Randy and Vadim
  • Concentrated gel purification sample from Sept. 16
  • Ligated ccdB gene into pSB1C3 vector
  • Transformed ccdB+pSB1C3 ligation into DB3 cells
  • Miniprepped ccdB+pSB1C3 ligation

• Samantha
  • PCR RD29A from pMA plasmid
  • Confirm PCR reaction via agarose gel

• Matthew
  • Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak
  • Sequences came back positive for EYFP and mCherry
    • Point mutation in the mCherry but shouldn't affect the translation

OCTOBER

Week of September 26-October 2:

• Christian
  • Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection
  • 5 colonies selected and inoculated overnight at 37C

• Elaine
  • Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission

• Randy and Vadim
  • Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst
    • Digest yielded successful ligation

• • Made Kan and Amp resistant plates
  • Made additional LB broth
  • Autoclaved labware and equipment
  • Miniprepped ligations from Sept. 23
  • Submitted ligation for sequencing at the Nevada Genomics Center
  • Made glycerol stock of ligations from Sept. 23
  • Transformed ligations from Sept. 23 into NEB cells
    • Plates yielded no growth-ccdB ligation successful

• Samantha
  • Gel purification of RD29A PCR product

• Matthew
  • Submitted Kozak-mCherry and Kozak-EYFP to iGEM
  • Cultured and miniprepped some of Elaine's GFP iGEM vector
  • Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo

Week of October 3-9:

• Christian
  • Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies
  • Digested results with EcoR1 and Pst1
  • Ran products on 0.8% agarose gel

• Expected bands show at ~550 bp
  • Sent samples for sequencing analysis
  • Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1
  • EtOH ppt of completed digest
  • T4 ligation (using custom buffer)
  • Digested Topocloned DREB1C_2 with EcoR1 and Spe1
  • Digested YFP_pSB1C3 with EcoR1 and Xba1
  • EtOH ppt of DREB1C/YFP digest products
  • T4 ligation (using custom buffer)
  • Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection

• Elaine
  • Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.

• The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp.
  • Topocloned Samantha Lee's PCR product #1 of rd29A
  • Streaked single colonies of rd29A (Topo Vector)
  • Cell cultured single colonies of rd29A (Topo Vector)
  • Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1.

• Matthew
  • Completed ligation and began transformation for 35S-GFP candidates
  • Screened 35S-GFP candidates on chloramphenicol and kanamycin
  • Cultured,miniprepped,digested, and ran on gel 20 candidates
    • One candidate possible for sequencing

 

35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right

From Left: Lane 1 - 1 kb ladder.

Lane 2 - Uncut 35S-GFP candidate 1

Lane 3 - 35S-GFP candidate 1

Lane 4 - Uncut 35S-GFP candidate 2

Lane 5 - 35S-GFP candidate 2

Lane 6 - Uncut 35S-GFP candidate 3

Lane 7 - 35S-GFP candidate 3

Lane 8 - Uncut 35S-GFP candidate 4

Lane 9 - 35S-GFP candidate 4

Lane 10 - 35S-GFP candidate 5

 

 

• • Submitted for sequencing 1 35S-GFP candidate

Week of October 10-16:

• Elaine
  • Ran rd29A (Topo Vector) gel.

• The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).
  • Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.
  • Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).
  • Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).
  • Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).

• Christian
  • Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection
  • 5 colonies selected and inoculated overnight at 37C
  • Miniprep 5 cultures and digested with EcoR1 and Pst1
  • Ran digests on 0.8% agarose gel

• Expected bands appeared at ~500 bp for sample 3 and sample 5
  • Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing
  • Sequencing data for DREB1C_2.3 had 0 mismatches and was selected

• Matthew
  • Sequencing results came back positive for 35S-GFP
  • Miniprepped 35S-GFP and made glycerol stock

• Richard and Nick
  • Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest

Week of October 17-23:

• Elaine
  • 30 Cell cultures of rd29A (pSB1C3)
  • Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested.
  • Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector
  • These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"

• The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.
• Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique and made sure I streaked the 45 single colonies on LB-Chloramphenicol plates.
  • Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels

• The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).
  • Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4) and streaked them on LB-Chloramphenicol plate.
  • Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer.
  • Sequenced Colony PCR #21 at the Nevada Genomics Center.
  • Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.
  • Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3.

• Christian
  • Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007
  • 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers
  • Products of PCR reaction run on a 0.8% agarose gel

• Band at ~500 bp for colony 24
  • Colony 24 was the only positive result
  • Colony 24 DREB1C/YFP construct was sent for sequencing

• Matthew
  • Submitted 35S-GFP to iGEM

• Richard and Nick
  • Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board

Week of October 24-30:

• Elaine
  • Added rd29A (pSB1C3) into the registry
  • Submitted rd29A (pSB1C3) through the mail.

 

 

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