iGEM 2010 — Team Nevada

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# Team:Nevada/Notebook

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* [Home](https://2010.igem.org/Team%3ANevada)
* [Team profile](https://2010.igem.org/Team%3ANevada/Team)
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  + [composite](https://2010.igem.org/Team%3ANevada/Composite)
  + [ccdB](https://2010.igem.org/Team%3ANevada/ccdB)
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  + [Results](https://2010.igem.org/Team%3ANevada/Results)
  + [Protocol](https://2010.igem.org/Team%3ANevada/BY-2%20%28NT1%29Transformation%20Protocol)
  + [Agro. Trans.](https://2010.igem.org/Team%3ANevada/Agrobacterium%20Transformations)
  + [Transgenic Plants](https://2010.igem.org/Team%3ANevada/Transgenic%20Plants)
* Other Stuff
  + [Modeling](https://2010.igem.org/Team%3ANevada/Modeling)
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  + [Notebook](https://2010.igem.org/Team%3ANevada/Notebook)
  + [Fundraising & Sponsorships](https://2010.igem.org/Team%3ANevada/fundraising_sponsorships)+ [Plant Summit](https://2010.igem.org/Team%3ANevada/Team%20Nevada%3A%20Plant%20Summit)
    + [Media](http://biotechniques.com/news/iGEM-competitors-gear-up-for-2010-challenge/biotechniques-304538.html)
    + [Cuckoo Clock](https://2010.igem.org/Team%3AFreiburg_Bioware/Team/Cuckoo_Clock)

Team Nevada Notebook

---

|  |
| --- |
| Contents   * [1 APRIL](#APRIL) * [2 MAY](#MAY) * [3 JUNE](#JUNE) * [4 JULY](#JULY) * [5 AUGUST](#AUGUST) * [6 SEPTEMBER](#SEPTEMBER) * [7 OCTOBER](#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

![PCR_ccdB_confirmation.png](https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png)

* + 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

[![Polasko eyfpcherry pcrreal.jpg](/wiki/images/a/ad/Polasko_eyfpcherry_pcrreal.jpg)](/File%3APolasko_eyfpcherry_pcrreal.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3APolasko_eyfpcherry_pcrreal.jpg "Enlarge")

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

[![GFP from iGEM (E0040) paint.jpg](/wiki/images/4/42/GFP_from_iGEM_%28E0040%29_paint.jpg)](/File%3AGFP_from_iGEM_%28E0040%29_paint.jpg)

* 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

[![GFP PCR product.jpg](/wiki/images/c/c3/GFP_PCR_product.jpg)](/File%3AGFP_PCR_product.jpg)

* 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

[![GFP in Topo Vector 001.jpg](/wiki/images/f/fb/GFP_in_Topo_Vector_001.jpg)](/File%3AGFP_in_Topo_Vector_001.jpg)

* 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

[![GFP in pSB1C3 iGEM Vector.jpg](/wiki/images/4/49/GFP_in_pSB1C3_iGEM_Vector.jpg)](/File%3AGFP_in_pSB1C3_iGEM_Vector.jpg)

* 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

[![DREB1C gel 1.jpg](/wiki/images/3/3f/DREB1C_gel_1.jpg)](/File%3ADREB1C_gel_1.jpg)

* 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

[![Polasko EYFPigem.jpg](/wiki/images/d/d4/Polasko_EYFPigem.jpg)](/File%3APolasko_EYFPigem.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3APolasko_EYFPigem.jpg "Enlarge")

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

[![Polasko mCherryigem.jpg](/wiki/images/b/bc/Polasko_mCherryigem.jpg)](/File%3APolasko_mCherryigem.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3APolasko_mCherryigem.jpg "Enlarge")

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

[![Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg](/wiki/images/8/8f/Rd29A_promoter_%2B_RFP_in_pSB1C3_iGEM_Vector.jpg)](/File%3ARd29A_promoter_%2B_RFP_in_pSB1C3_iGEM_Vector.jpg)

* 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

![CcdB%2BpSB1C3_gel.png](https://static.igem.org/mediawiki/2010/b/bf/CcdB%2BpSB1C3_gel.png)

* + 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

[![DREB1C gel 2.jpg](/wiki/images/b/bd/DREB1C_gel_2.jpg)](/File%3ADREB1C_gel_2.jpg)

* 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.

[![Rd29A PCR product - Samantha Lee.jpg](/wiki/images/6/66/Rd29A_PCR_product_-_Samantha_Lee.jpg)](/File%3ARd29A_PCR_product_-_Samantha_Lee.jpg)

* 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

[![Polasko 35sgfp.jpg](/wiki/images/7/7d/Polasko_35sgfp.jpg)](/File%3APolasko_35sgfp.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3APolasko_35sgfp.jpg "Enlarge")

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.

[![Rd29A in TOPO Vector.jpg](/wiki/images/a/ac/Rd29A_in_TOPO_Vector.jpg)](/File%3ARd29A_in_TOPO_Vector.jpg)

* 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

[![DREB1C gel 3.jpg](/wiki/images/3/31/DREB1C_gel_3.jpg)](/File%3ADREB1C_gel_3.jpg)

* 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"

[![Rd29A in pSB1C3 - Epic Failure.jpg](/wiki/images/b/b0/Rd29A_in_pSB1C3_-_Epic_Failure.jpg)](/File%3ARd29A_in_pSB1C3_-_Epic_Failure.jpg)

* 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

[![Colony PCR -21 - rd29A in pSB1C3 Vector.jpg](/wiki/images/4/48/Colony_PCR_-21_-_rd29A_in_pSB1C3_Vector.jpg)](/File%3AColony_PCR_-21_-_rd29A_in_pSB1C3_Vector.jpg)

* 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

[![DREB1C gel 4.jpg](/wiki/images/0/03/DREB1C_gel_4.jpg)](/File%3ADREB1C_gel_4.jpg)

* 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.

---

| [![Nevada CABNR.jpg](/wiki/images/d/de/Nevada_CABNR.jpg)](/File%3ANevada_CABNR.jpg) | [![NV INBRE Logo.jpg](/wiki/images/d/d7/NV_INBRE_Logo.jpg)](/File%3ANV_INBRE_Logo.jpg) | [![UNR ASUN logo.jpg](/wiki/images/9/95/UNR_ASUN_logo.jpg)](/File%3AUNR_ASUN_logo.jpg) | [![Promega logo.jpg](/wiki/images/a/ab/Promega_logo.jpg)](/File%3APromega_logo.jpg) | [![Invitrogen logo.jpeg](/wiki/images/5/56/Invitrogen_logo.jpeg)](/File%3AInvitrogen_logo.jpeg) | [![Sda logo small.jpg](/wiki/images/0/0d/Sda_logo_small.jpg)](/File%3ASda_logo_small.jpg) |
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# Team:Nevada/Results

### From 2010.igem.org

[![Results UNR Final.png](/wiki/images/2/2b/Results_UNR_Final.png)](/File%3AResults_UNR_Final.png)

* [Home](https://2010.igem.org/Team%3ANevada)
* [Team profile](https://2010.igem.org/Team%3ANevada/Team)
* Project
  + [Remote Sensors](https://2010.igem.org/Team%3ANevada/plants_as_remote_sensors)
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* Parts
  + [promoters](https://2010.igem.org/Team%3ANevada/promoters)
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  + [composite](https://2010.igem.org/Team%3ANevada/Composite)
  + [ccdB](https://2010.igem.org/Team%3ANevada/ccdB)
* [Registry Submissions](https://2010.igem.org/Team%3ANevada/registry_submissions)
* [NT Cell Transformations](https://2010.igem.org/Team%3ANevada/Transformations)
  + [Results](https://2010.igem.org/Team%3ANevada/Results)
  + [Protocol](https://2010.igem.org/Team%3ANevada/BY-2%20%28NT1%29Transformation%20Protocol)
  + [Agro. Trans.](https://2010.igem.org/Team%3ANevada/Agrobacterium%20Transformations)
  + [Transgenic Plants](https://2010.igem.org/Team%3ANevada/Transgenic%20Plants)
* Other Stuff
  + [Modeling](https://2010.igem.org/Team%3ANevada/Modeling)
  + [Safety](https://2010.igem.org/Team%3ANevada/Safety)
  + [Notebook](https://2010.igem.org/Team%3ANevada/Notebook)
  + [Fundraising & Sponsorships](https://2010.igem.org/Team%3ANevada/fundraising_sponsorships)+ [Plant Summit](https://2010.igem.org/Team%3ANevada/Team%20Nevada%3A%20Plant%20Summit)
    + [Media](http://biotechniques.com/news/iGEM-competitors-gear-up-for-2010-challenge/biotechniques-304538.html)
    + [Cuckoo Clock](https://2010.igem.org/Team%3AFreiburg_Bioware/Team/Cuckoo_Clock)

## NT Cell Transformation Results

# Subpages

* [Results](https://2010.igem.org/Team%3ANevada/Results)
* [NT Cell Transformation Protocol](https://2010.igem.org/Team%3ANevada/BY-2%20%28NT1%29Transformation%20Protocol)
* [Agrobacterium Transformations](https://2010.igem.org/Team%3ANevada/Agrobacterium%20Transformations)
* [Transgenic Plants: into the Wild](https://2010.igem.org/Team%3ANevada/Transgenic%20Plants)

[![](https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG)](https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG)
**Plated NT Cells Immediately after Transformation**

---

[![](https://static.igem.org/mediawiki/2010/6/60/Igem_ntcells(2).jpg)](https://static.igem.org/mediawiki/2010/6/60/Igem_ntcells%282%29.jpg)
**Plated Transformed NT Cells after about 3 weeks of Cell Growth**

---

[![](https://static.igem.org/mediawiki/2010/9/99/Rd29A_results.png)](https://static.igem.org/mediawiki/2010/9/99/Rd29A_results.png)

**rd29A + RFP Transformed NT Cells after about 3 weeks of growth:** The NT cells shown above were transformed with rd29A+RFP, a composite part in which the rd29A promoter is turned on due to external stimuli such as cold, drought or salinity. When the rd29A promoter is turned "on" it then turns on its reporter, in this case RFP. After about 3 weeks of NT cell growth, the transformed cells were "cold stressed" for about 3hrs in 4 degrees Celsius. The results shown indicate that when cold stressed, the transformed NT cells become fluorescent with RFP. Over the next few weeks we hope to see this signal grow stronger and test these NT cells' responsiveness to other stresses that induce the rd29A promoter.

---

**35S + GFP Transformed NT Cells:** Currently, our transformed NT cells that contain the composite part, 35S + GFP, have not grow enough to determine whether or not the transformation was successful. The 35S promoter was designed as a constitutive control and would always be turned "on", thus maintaining a constant GFP fluorescent signal. Over the next few weeks we hope to see more cell growth and eventually transformed cells that produce green fluorescent protein constitutively.

---

| [![Nevada CABNR.jpg](/wiki/images/d/de/Nevada_CABNR.jpg)](/File%3ANevada_CABNR.jpg) | [![NV INBRE Logo.jpg](/wiki/images/d/d7/NV_INBRE_Logo.jpg)](/File%3ANV_INBRE_Logo.jpg) | [![UNR ASUN logo.jpg](/wiki/images/9/95/UNR_ASUN_logo.jpg)](/File%3AUNR_ASUN_logo.jpg) | [![Promega logo.jpg](/wiki/images/a/ab/Promega_logo.jpg)](/File%3APromega_logo.jpg) | [![Invitrogen logo.jpeg](/wiki/images/5/56/Invitrogen_logo.jpeg)](/File%3AInvitrogen_logo.jpeg) | [![Sda logo small.jpg](/wiki/images/0/0d/Sda_logo_small.jpg)](/File%3ASda_logo_small.jpg) |
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