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Contents 1 6/14-18/10 (Boot Camp) 2 6/22/10 3 6/23/10 4 6/24/10 5 6/25/10 6 6/28/10 7 6/29/10 8 6/30/10 9 7/1/10 10 7/2/10 11 7/3/10 12 7/4/10 13 7/5/10 14 7/6/10 15 7/7/10 16 7/8/10 17 7/9/10 18 7/10/10 19 7/11/10 20 7/12/10 21 7/13/10 22 7/14/10 23 7/15/10 24 7/16/10 25 7/19/10 26 7/20/10 27 7/21/10 28 7/22/10 29 7/23/10 30 7/26/10 31 7/27/10 32 7/28/10 33 7/29/10 34 7/30/10 35 8/2/10 36 8/3/10 37 8/4/10 38 8/5/10 39 8/6/10 40 8/9/10 41 8/10/10 42 8/11/10 43 8/12/10 44 8/13/10 45 8/16/10 46 8/17/10 47 8/18/10 48 8/19/10 49 8/20/10 50 8/21/10 51 8/22/10 52 8/23/10 53 8/24/10 54 8/25/10 55 8/26/10 56 8/27/10 57 8/30/10 58 8/31/10 59 9/1/10 60 9/2/10 61 9/3/10 62 9/6/10 63 9/7/10 64 9/8/10 65 9/9/10 66 9/10/10

6/14-18/10 (Boot Camp)

DNA extracted from kit

Part: [http://partsregistry.org/partsdb/get_part.cgi?part=BBa_I746200]

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6/22/10

We poured our Kan, Amp, Tet, Kan+Amp, Kan+Tet, Amp+Tet antibiotic agar plates.

Antibiotic Concentrations

• Amp: 50mg/500ml

• Kan: 31.97mg/500ml

• Tet: 6mg/500ml

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6/23/10

Resuspended 12O and 6G DNA from the iGEM Kit and transformed them into our competent cells.

• 6G = r0011(inducible promoter)
• 12O = e0840(rbs30-gfp-2xterm)

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6/24/10

Ran a gel of our PCR product to make sure that our taq polymerase master mix was working.

Selected single 12O and 6G colonies and transferred them to LB and incubated for 18 hours.

Let 1 12O and 1 6G plate incubate at RT. Let 1 12O and 1 6G plate incubate at 37°C. Intended to observe lawn growth.

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6/25/10

Low DNA yield; Plan to redo DNA extraction from Kit

Found red/pink colonies in 12O plates. Thought to be contaminations. Plates were thrown out.

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6/28/10

Kit to Stock

• 1-12O (e0840: rbs30-gfp-2xterm)

• 1-6G (r0011: inducible promoter)

• 3-20M (K124017: Bacteriophage Lysis Cassette S105, R, and Rz)

Plates were incubated at 37°C overnight (starting at 2:30 pm).

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6/29/10

Selected single 1-12O, 1-6G, 3-20M colonies and grew them in Amp-LB broth for 18 hours (starting at 4:15 pm).

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6/30/10

Poured 2 Sleeves of Kan and Amp plates respectively

Met with Dr. Russin (BIF)

Sent emails regarding biofilm and biofilm imaging: Aaron Packman, Wei Zhang, Kimberly Grey

Most likely going to use water immersion Leica (Confocal Microscope) + low MP agar (enrobing technique)

Red colonies reappeared on 1-12O plates; Fluorescent Microscopy revealed negligible activity; still unsure why colonies are red.

Miniprepped 1-12O, 3-20M, 1-6G: nanodrop -> mostly below 20ng, 1 above 100ng; stored in -20°C

Re-miniprepped after growing in liquid LB for 3 hours; nanodrop -> around 50ng/μL, 2 around 100ng/μL; stored in -20°C

Transferred overnight cultures into new 5ml LB broths. Incubated overnight in an angled shaker (starting from 4:20pm)

Plated 10μl, 25μl, 100μl, and 200μl (added LB up to 200μL) of the KR 1-12O overnight culture. Intend to measure the thickness of E.Coli lawn.

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7/1/10

First weekly meeting with Professor Jewett, Leonard, and Mordacq.

Removed overnight cultures from incubator at 10:00am. Miniprep (let the elution buffer sit for 10 minutes with the DNA before centrifuging) --> 1 around 30ng/μL, mostly around 75ng/μL.

Started PCR of the chitin synthase gene using yeast genome.

Kit to Stock:

• 1-12O (e0840: rbs30-gfp-2xterm)
• 1-12M (e0240: rbs32-gfp-2xterm)
• 1-6G (r0011: inducible promoter)

Plates were incubated at 37°C overnight (starting at 5pm).

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7/2/10

Removed overnight plates from 37°C at 10:00am and moved them to the 4°C cold room.

Gel extraction on Chitin Synthase PCR product.

Colonies were selected from the NB-12M, NB-6G, NB-12O, T10-12M, T10-6G, and T10-12O plates and transferred to LB broth and incubated for 18 hours at 37°C (starting at 4:00pm).

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7/3/10

Miniprep -> ~50ng/μl

Called Avi about kit to stock protocol - "18hr way too long"

2 Sets of colonies were selected from the NB-12M, NB-6G, NB-12O, T10-12M, T10-6G, and T10-12O plates and transferred to LB broth and incubated for 13 and 15 hours at 37°C (starting at 9:15pm).

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7/4/10

13hr(2ml) -> miniprep -> ~45ng/μl

13hr(3ml) -> miniprep -> ~55ng/μl

15hr(2ml) -> miniprep -> 40ng/μl

Also, NB consistently gave higher yields than T10 by 5~10ng/μl

Note: 18hr gave higher yields than either 13hr or 15hr

Protocol: 18hr, NB, 5ml incubation (spin all down)

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7/5/10

CHS3 Gel Extraction -> 8ng/μl

Restarted CHS3 PCR added 5 cycles.

Kit to Stock:

• 2-8E (j06702: RFP)
• 1-18C (j23100: CP1)
• 1-18K (j23104: CP2)
• 1-18M (j23105: CP3)
• 1-2M (b0034: RBS1)
• 1-2G (b0031: RBS2)
• 1-2I (b0032: RBS3)
• 1-23L(b0015: DT1)
• 1-2O (c0012: LacL)

Reincubated (5μl transfer to liquid culture) 1-12O, 1-12M, 1-6G (GFP1, GFP2, LacP1 respectively)

Notes: Need Cells; Reorganize Lab (assign benches)

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7/6/10

Miniprep (16hr/5ml) -> around 50ng/μl

Ran another gel extraction for CHS3

Kit to Stock:

• 1-1D (r0010: LacP2)
• 3-20K (K124014: Holin2)

Plated 1-1D, 3-20K and incubated overnight.

Colonies were selected from the NB-8E, NB-18C, NB-18K, NB-18M, NB-2M, NB-2G, T10-2I, T10-23L, T10-6K, and T10-2O plates and transferred to LB broth and incubated for 16 hours at 37°C (starting at 6:00pm).

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7/7/10

Miniprep (25ul of Amp/5ml spin down) --> 3 ~100ng/μl and 2 ~180ng/μl

Miniprep (50ul of Amp/1.5ml spin down) --> all ~20ng/μl

Miniprep (50ul of Amp/3.5ml spin down) --> all ~20ng/μl

50μg/ml of Amp = seems to result in more DNA (half the concentration of what we previously used)

Gel extraction --> very bright CHS3 band (10.1ng/μl)

Colonies were selected from the T10-2I, T10-23L, T10-6K, T10-2O, T10-1D, T10-20K plates and transferred to LB broth and incubated for 16 hours at 37°C (starting at 7:30pm)

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7/8/10

Ethanol precipitation of 1-6G, 2-8E, 1-12O, and 1-12M

Keio Collection: ChiA knockouts grew, but YdgG knockouts did not (possibly dead due to storage in -20 instead of -80)

Started another PCR reaction of CHS3

Miniprep (5ml spin down/25ul of antibiotic) --> mostly ~50ng/ul

Miniprep (5ml spin down/20ul of antibiotic) --> mostly ~70ng/ul

Diagrammed out our assembly methods for the CHS3 plasmid and the Apoptotic plasmid

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7/9/10

Finished the ethanol precipiation (RESULTS)

Started assembling the LacP-RFP, LacP-GFP1, and LacP-GFP2 pieces for use in testing the Lac promoter.

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7/10/10

IPTG Spray Bottles: 10 Sprays = ~1.25ml --> .125ml/spray 100mM IPTG Stpcl = 1 gram/41.96ml

3 Sprays of 1mM IPTG (started at 5:15pm)

• 30 Min
• 1 Hour

3 Sprays of 5mM IPTG (started at 5:15pm)

• 30 Min
• 1 Hour

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7/11/10

Roughly 60% of 1-12O colonies expressed green fluorescence Only a few colonies of 1-12M expressed green fluorescence No red fluorescence was seen.

Miniprepped DNA (LacP1-GFP construct) --> stored in -20°C

IPTG seems unnecessary for expression of GFP via the LacP promoter - very leaky --> need LacL repressor

Ran PCR products in gel -> good yield

Kit to Stock:

• 1-18I (e0430: YFP)
• 1-18F (e1010: RFP)
• 1-20L (q001121: LacPI1)
• 1-20P (q04121: LacPI2)
• 1-10H (i712019: Luciferase)
• 1-3A (psb1c3: BBPlasmid-C)
• 1-5A (psb1k3: bbplasmid-K)
• 1-7A (psb1T3: bbplasmid-T)
• 1-1C (psb1A3 bbplasmid-A)

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7/12/10

All backbone plasmid transformation colonies appear reddish/purple (due to RFP gene in plasmids)

Colonies were selected from the yfp (e0430 1-18I), rfp (E1010 1-18F), lacpl1 (Q001121 1-20L), lacpl2 (Q04121 1-20P), luciferase (I712019 1-10H), bbplasmid-c (psb1c3 1-3A), bbplasmid-k (psb1k3 1-5A), bbplasmid-t (psb1T3 1-7A), bbplasmid-A (psb1A3 1-1C), 1-2I, and 2-6K plates and incubated in LB broth at 37C for 16 hours (starting at 6:00pm)

Received sponsorship from NEB.

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7/13/10

Miniprep --> mainly ~200ng/ul (3 parts that we redid were still low = ~50ng/ul)

Used 3A assembly to assemble LacP1-GFP, LacP2-GFP, and LacPI1-GFP (in 4 different backbone plasmids, psb1A3/psb1T3/psb1C3/psb1K3, made from transforming from well parts)

Met with Wei Zhang to discuss biofilm growth. Decided flow cells would not be the most cost effective and useful way to grow our E.Coli biofilm. Plan on using normal agar plates. Discovered that BacLight Live/Dead assay can be used on bacteria grown on a plate rather than bacteria growth in broth. We were told to add the BacLight dye to the lawn of bacteria and allow for it to diffuse before washing with ______.

Received sponsorship from Promega.

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7/14/10

None of the assemblies grew.

Group Meeting:

• Need to keep notes for future team (i.e. sponsorships, changes to protocol, etc.)
• Made a list of things to get from Promega

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7/15/10

PCR Purified CHS3 PCR product

Ran 3 PCR reactions using Amp, Chl, Tet linearized backbone plasmids as the template

3A Assembly of CP1-GFP, LacP1-GFP, LacP2-GFP, LacPI1-GFP, LacPI2-GFP, and CHS3-XX.

Streaked TOP10 cells on agar plates and incubate overnight for making competent cells.

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7/16/10

None of the assemblies grew.

PCR Purified backbone plasmid PCR products.

3A Assembly of CP1-GFP, LacP-GFP, LacPI1-GFP.

Selected single TOP10 colonies and prepared seed stocks for making competent TOP10 cells.

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7/19/10

None of the assemblies grew.

Had 2 different team members retry 3A assembly of CP1-GFP, LacP-GFP, LacPI1-GFP. One team member did not purify digestion products. One team member PCR purified digestion products.

Ran a gel of the digestion products --> restriction digest was successful

Inoculated TOP10 seed stocks for 2 hours 37C --> OD600nm of 0.25 Made the TOP10 cells competent using the OpenWetWare protocol.

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7/20/10

None of the assemblies grew.

Ran a gel of the ligation products --> ligation was successful, but very little DNA

Tried 3A assembly using 50μl of competent TOP10 cells with either 1μl, 5μl, or 10μl of ligation product.

Ethanol precipitation of 1-2I, 1-6G, and 2-6K.

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7/21/10

Transformed and plated TOP10 competent cells using a standard plasmid --> plan to test competency tomorrow

Poured 1 sleeve of Amp plates.

We came to the conclusion that our old Tet plates either had too little Tet antibiotic or the antibiotic had degraded from exposure to light --> poured 1 sleeve of Tet plates

Transformed 5μl of LacPI1-GFP and CP1-GFP ligation product with 25μl of competent TOP10 cells.

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7/22/10

Weekly meeting with advisers. Went over MAGE knock-out technique. Discussed our problems with 3A assembly.

Measured the competence of TOP10 cells --> 9600 cfu/μgDNA

Transformed TOP10 competent cells using pUC19 standard plasmid DNA. Plan to measure competence again tomorrow.

Streaked EcNR2 cells on Chl and Carbenicillin. Plan to use this strain carry out our MAGE double knock-out.

3A Assembly of CP1-GFP, LacP-GFP, LacPI1-GFP. Ran 2 parallel assemblies - one with a 10 min ligation step and one with a 1 hour ligation step. Transformed using both TOP10 cells and BL21-Gold cells.

Kit to stock of 1-2I and 2-6K.

Received sponsorship from EMD Chemicals through Millipore.

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7/23/10

Realized that Chl was never added to our Chl plates --> added 200ul of 1x Chl solution to our old Chl plates to salvage the plates

Poured 2 new sleeves of Chl plates.

Ran a gel of Chl and Tet linear plasmid backbone PCR product --> successful PCR

PCR purified Chl and Tet linear plasmid backbone PCR product.

Plated Gold 2ul-ligation product 10min-ligation time (CP1-GFP), Gold 2ul-ligation product 1hr-ligation time (LacP1), Gold 2ul-ligation product 10min-ligation time (LacPI1), Gold 8ul-ligation product 1hr-ligation time (CP1-GFP), Gold 8ul-ligation product 10min-ligation time (LacP1), and Gold 8ul-ligation product 1hr-ligation time (LacPI1) transformed cells.

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7/26/10

BIF confocal microscopy training

Kit to stock of 1-6G and 1-12O

Ran a gel of the pMAL PCR products --> very faint bands

Used pMAL PCR products as template for another PCR reaction

Ligated LacP1-GFP and transformed using our own TOP10 competent cells

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7/27/10

Excessive growth of 1-6G and 1-12O plates --> amp plates are possibly bad

Gel extraction LacP1, GFP, Linear Tet backbone plasmid restriction digest products

Ran a gel of digestion and ligation products; conclusion: linear plasmid PCR products are bad. Also threw out other assortment of degraded DNA.

3A Assembly using 1 ug of starting DNA.

Poured new Tet and Chl plates

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7/28/10

Followed Knight's 3A Assembly for LacP1-GFP part --> No growth

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7/29/10

3A Assembly using Knight protocol restriction digests --> but replaced linear backbone plasmid with circular plasmid miniprepped from IGEM kit

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7/30/10

LACP1 with GFP in CP-C yielded good results

Ligations (CP1, LACP1, LACPI1 with GFP1 in CP-C)

Redid PCR of CHS3 for pMAL insert

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8/2/10

Competency test (iGEM T10 from Leonard T10) -->

cP 3A Assembly troubleshooting: why are there white colonies? --> Ran Gel along with Red and Green (lacp-gfp in cpc) (Sequencing necessary?)

1050 (rfp default insert), 2150 (bbp), 55+878 (lacp,gfp)

red should be - 3200, 1050, 2150

white should be - 2150

green should be - 3083, 933, 2150

cP 3A Assembly troubleshooting: How to improve efficiency **TRY PHOSPHATASE IN PARALLEL

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8/3/10

Gel Results (White, Green, Red Colonies from 3A Assembly)

• Red colonies look normal
• Green colonies look normal
• White colonies --> abnormal bands at 750bp, 1750bp, 2600bp, and 4300bp

The white colonies could be due to aggregation of GFP or RFP protein due to excessive amounts of plasmid.

Inoculated CP1-LacPi1, CP2-LacPi1, CP3-LacPi1, CP1-LacPi2, CP2-LacPi2, CP3-LacPi2, Holin1-XX, Holin2-XX, PMAL, GFP, LacPi1, LacPi2, YdgG Knockout, ChiA Knockout.

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8/4/10

Miniprepped CP1-LacPi1, CP2-LacPi1, CP3-LacPi1, CP1-LacPi2, CP2-LacPi2, CP3-LacPi2, Holin1-XX, Holin2-XX, PMAL, GFP, LacPi1, LacPi2.

Miniprepped (CP1-Lacpi1) part --> digested with E/P --> ran a gel

• Should be 35+1372/1370 = 1405/1407 +20(pre/suffix)

Miniprepped (Holin1-XX) and (Holin2-XX) part --> digested with E/P ran a gel

• Should be 1257, 317 + 129 = 1386, 446 +20 (prefix/suffix)

Made glycerol stock of ________

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8/5/10

Made competent ChiA knockout cells

Made electrocompetent ECNR2 cells

Minipreppred (pMal-CHS3) part

Assembled and transformed (CP1-LacPI1)-(GFP), (CP2-LacPI1)-(GFP),(CP3-LacPI1)-(GFP),(CP1-LacPI2)-(GFP),(CP2-LacPI2)-(GFP), (CP3-LacPI2)-(GFP)

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8/6/10

Lysed and stained (pMal-CHS3) cells with calcofluor white --> for chitin

Transformed ChiA knockout competent cells with _____ --> plan to test competency

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8/9/10

Bought Chitosan powder to use as a positive control for calcofluor white --> glowed bright blue/green when stained

Stained (pMal-CHS3) ligation products for the presence of chitin --> ligation 1/tube 3 had slight fluorescence

Used UV-vis Spectroscopy to measure chitin concentrations in ________

Grew bakers yeast to test for presence of chitin

Colony PCR on (pMal-CHS3)

CP-LacPI part testing

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8/10/10

Ran gel for colony PCR no positives

Ran PCR for CHS3 insert into pMal vector

Stained varying concentrations of chitosan (.25g/ml, 25ug/ml, 5ug/ml, 1ug/ml) and (pMal-CHS3) cells --> cells showed no signs of chitin

Digested __________ (sean)

Ligated (CP-LacPi)-(GFP) in Tet backbone

Made LB minimum media for electroporated cells

Miniprepped circular backbone plasmid

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8/11/10

Digested and ran a gel of CHS3 and backbone plasmids (C,T,K,A)

Tested restriction enzymes by digesting circular plasmids with each enzyme individually --> should see 1 2500kb band

Organized the DNA samples in our -20.

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8/12/10

Weekly meeting

• Went over our gels --> identified faulty parts (holin1, holin2, CHS3-pMal, CP-LacPI)
• Plan to re-kit to stock and reassemble

Decided to use an alternative stain because calcofluor results in too much background and our confocal microscope can not excite in the UV spectrum

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8/13/10

Methanol fixation of yeast and E.Coli cells --> stained with rhodamine-conjugated chitin probe (allowed to incubate overnight)

Competency of ChiA knockouts = 2.5x10^7 and 5.0x10^6

Cycles 1 of File:MAGE for tqsA Knockouts.

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8/16/10

Cloned CHS3 into Chlor Biobricks plasmid --> will be used for Site Directed Mutagenesis

Cycles 2 and 3 of MAGE for tqsA Knockouts.

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8/17/10

Site-Directed Mutagenesis to remove the PstI site in CHS3

Cycles 4-6 of MAGE for tqsA Knockouts.

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8/18/10

Site-Directed Mutagenesis

Cycle 7 of MAGE for tqsA Knockouts.

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8/19/10

Site-Directed Mutagenesis

Cycles 8 and 9 of MAGE for tqsA Knockouts.

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8/20/10

Site-Directed Mutagenesis-Redo PCR. 10 colonies were screened, all negative (Two bands were seen when cut with PstI)

Cycles 10-12 of MAGE for tqsA Knockouts.

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8/21/10

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8/22/10

Cycle 13 of MAGE for tqsA Knockouts.

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8/23/10

Site-Directed Mutagenesis

Cycles 14 and 15 of MAGE for tqsA Knockouts.

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8/24/10

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8/25/10

Regrew Cycle 15 MAGE cells in liquid culture.

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8/26/10

Screened MAGE culture for knockouts.

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8/27/10

Screened MAGE culture for knockouts.

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8/30/10

Screened MAGE culture for knockouts.

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8/31/10

Attempted to assemble LacP-RBS-CHS3 --> accidentally cut CHS3 with P (contains a P site)

Reorganized the racks in the -20.

Threw out unnecessary/old DNA from our -20.

Moved all of our racks from our -20 to another -20 in order to defrost our -20.

tqsA Knockouts FOUND (?) in MAGE culture upon screening.

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9/1/10

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9/2/10

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9/3/10

Reorganized -20 DNA stock. Created racks for individual teammates to stay organized once the school year starts.

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9/6/10

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9/7/10

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9/8/10

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9/9/10

Assembled LacP-RBS1-CHS3 in a Chlor backbone plasmid using standard assembly methods Cut LacP-RBS1 using S Cut CHS3 using X and S (50% chance of inserting backwards --> must select for this after)

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9/10/10

Inoculated 20 LacP-RBS-CHS3 cells.

Assembled CP-LacPI-RFP parts into Tet backbone.

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