Team:Northwestern/Notebook

Brainstorming April-June 2010

Week1 6/13/10-6/19/10

Week2 6/20/10-6/26/10

Week3 6/27/10-7/3/10

Week4 7/4/10-7/10/10

Week5 7/11/10-7/17/10

Week6 7/18/10-7/24/10

Week7 7/25/10-7/31/10

Week8 8/1/10-8/7/10

Week9 8/8/10-8/14/10

Week10 8/15/10-8/21/10

Week11 8/22/10-8/28/10

Week12 8/29/10-9/4/10

Week13 9/5/10-9/11/10

Week14 9/12/10-9/18/10

Week15 9/19/10-9/25/10

Week16 9/26/10-10/2/10

Week17 10/3/10-10/9/10

Week18 10/10/10-10/16/10

Week19 10/17/10-10/23/10

Week20 10/24/10-10/30/10

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

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)

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.

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.

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).

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).

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.

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).

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).

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).

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)

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)

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).

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)

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

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.

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

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)

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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

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

7/28/10

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

7/29/10

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

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

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

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.

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 ________

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)

8/6/10

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

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

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

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

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.

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

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.

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.

8/17/10

Site-Directed Mutagenesis to remove the PstI site in CHS3

Cycles 4-6 of MAGE for tqsA Knockouts.

8/18/10

Site-Directed Mutagenesis. Ran screening for 8/17, none were positive so repeated reaction with better protocol. (PCR only)

Cycle 7 of MAGE for tqsA Knockouts.

8/19/10

Site-Directed Mutagenesis. Transformed products from 8/18

Cycles 8 and 9 of MAGE for tqsA Knockouts.

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.

8/21/10

8/22/10

Cycle 13 of MAGE for tqsA Knockouts.

8/23/10

Site-Directed Mutagenesis

Cycles 14 and 15 of MAGE for tqsA Knockouts.

8/24/10

Ligated CP-LacPI-RFP combinations.

8/25/10

Regrew Cycle 15 MAGE cells in liquid culture.

8/26/10

Screened MAGE culture for knockouts.

8/27/10

Screened MAGE culture for knockouts.

8/30/10

Screened MAGE culture for knockouts.

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.

9/1/10

Ragan left for home today and left MAGE in our hands. He asked us to do the following tomorrow:

1. In the shaking 30C incubator, there are two tubes. One has what appear to be our knockout strain, while the other is a control and should have no growth. If there is cell growth in the control tube, then the "MAGE only" media with lesser volume is contaminated and must be thrown out. The strain of interest is colony V in the small A+C plate marked with a giant asterisk on Matt's shelf in the 4C. In the future, if you need to use the unopened container of MAGE only media, you must put Amp and Chlor in it first to prevent contamination. If there is growth in the tqsA knockout tube and none in the control strain, plate the knockouts in a 1:10-6 dilution on an A+C plate, grow this at 30C, and move the culture tube itself to the 4C.
2. In the 37C incubator, there should be two Amp plates labeled as Puc electro. These should have colonies. Let me know if they do not.
3. In the non-shaking 30C incubator, there should be 3 EcNR2 plates. Save these for now; once we know beyond a shred of doubt that the knockouts are real, we can throw these out.
4. Run the following PCR reaction:

Make two mixes:

1 ul Taq buffer (red box)

1 uL dNTPs (use the tube labeled dNTPs RP - this is a 1:4 dilution from Ben's dNTPs)

0.15 uL 40 mM Forward Primer for one, 0.30 uL for the other

0.15 uL 40 mM Reverse Primer for one, 0.30 uL for the other

6.6 uL Nuclease Free Water

Pick ANY non-MAGE strain. Pick it with a pipet tip and swirl it in each of these reactions. Use two different tips.

Put this in the right thermocycler under the program tqsA. After the first 3 minutes of 95C have passed, pause the machine, open the top and quickly pipet 0.10 uL of Taq Polymerase (also in the red box) into each tube. Be sure to use two different tips. Close the top and resume the program.

Run this with the NEB 100 bp ladder (we should make aliquots of this and freeze the rest to prevent degradation) in a 2% gel at 90-100 volts.

When you read this gel, you should see NO BAND at 340 bp. If you do, this likely means that the forward primer isn't specific enough to the knockout. This is a control. If we see no band, then the tqsA knockouts are true knockouts.

After all this is done, someone should continue with MAGE from the tqsA knockouts for the chiA knockouts. I showed Kevin how to do it, but if anyone has any questions let me know. A copy of the protocol is on the bench in the 4C. In fact, I'd rather whoever decides to do it talk to me ahead of time to make sure everything is clear. For instance, the cuvettes will need to be washed somewhere in the process since we won't have enough.

9/2/10

9/3/10

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

9/6/10

Kevin did some reorganizing to get the lab back in order for Prof. Mordacq's class this fall. He consolidated the bench space, the -20 refrigerator and the cold room. Thanks!

9/7/10

9/8/10

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)

9/10/10

Inoculated 20 LacP-RBS-CHS3 cells.

Assembled CP-LacPI-RFP parts into Tet backbone.

9/13/10

Assembled LacP-RBS-CHS3 using standard assembly. Grew on Chlor plates.

9/14/10

Inoculated Apop1

Inoculated 18 colonies of LacP-RBS-CHS3 for screening purposes

Plated CHS3,CP-LacPI-GFP, CP-LacPI-RFP.

9/15/10

Miniprepped LacP-RBS-CHS3 and Apop1

Digested LacP-RBS-CHS3 with X and P (CHS3 contains an internal P site)

Kevin put 15 overnight cultures into the 37 degree incubator at 6pm.

Kevin the CP-LACPI assemblies as well as the CP-LACPI-GFP/RFP assemblies around 9 pm. There was one normal size KAN plate for each CP-LACPI-GFP/RFP assembly (including a negative control) and two small size TET plates for each CP-LACPI assembly (with only one negative control plate).

Timi poured two sleeves of mini-Chlor and one sleeve of mini-Tet plates.

9/16/10

Ran digested LacP-RBS-CHS3 on a gel:

• Majority contained 1 band around 3600bp
• 3 contained 3 bands (2000bp, 1600bp, 1100bp)

Timi mini-prepped the 15 overnight cultures that Kevin put into the incubator last night.

9/17/10

Digested LacP-RBS(1), LacP-RBS(2), and LacP-RBS(3) with ES --> Ran on a gel LacP-RBS(2) and LacP-RBS(3) showed correct bands at ~70bp

Timi miniprepped the cultures Kevin inoculated last night. Note: the tube caps were snapped shut while they were shaking-- that probably affected the DNA concentration of some of the minipreps.

Anyhow- results! Two of the cultures did not have any cells so he probably just missed the colony or something- not a big deal.

CP3-LacPi2 retrans: 1) 155.3 2) 135.1 3) 76.8

CP1-LacPi2 seq: 1) 102.9 2) 91.7 3) 71.7

CP2-LacPi2: 1) 83.8 2) 89.6

CP1-LacPi2 1) 54.7 2) 94.7

CP1-LacPi1: 1) 108.8 2) 66.0 3) 36.5

The concentrations are all on the lower side compared to what we have been getting. I would say to just send some out for sequencing to see if the ligations actually worked before worrying about the concentrations. These DNA vials are in my DNA box in the -20.

PLATES:: The CP-LACPI assemblies as well as the CP-LACPI-GFP/RFP assembly plates still had really small colonies on them. Timi came back later to put them into the cold room.

Kevin put in another series of CP-LacPI-GFP/RFP incubations at around 6pm.

9/20/10

iGEM abstracts were due tonight. We had an after-hours lab meeting to finish things up. Prof. Leonard stopped by to help us make some final edits and to figure out how much money we had left in the budget.

We also took this time to catch eachother up on what we had been working on since team members started leaving for home.

9/21/10

Timi and Kevin poured a large gel prior to heading home for the night.

9/22/10

Timi and Kevin loaded digested CP-LacPI assemblies into gels this morning and Kevin ran the gel between classes.

9/23/10

Early 8am team meeting with Prof. Mordacq.

CHS3 made, low conc (25-35) 5 tubes of about 80ul each in 4degree

9/24/10

Digested LacP-RBS(2) with ES. Digested Tet plasmid with ES.

9/25/10

We had a group lab meeting to begin working on the poster and presentation.

Timi and Kevin put 18 overnight cultures of CP-LacPI assemblies into the incubator with Tet antibiotic (UHOH!).

The Wildcats won their game! 4-0! Go cats! :)

9/26/10

Upon taking out the cultures, Kevin realized that he and Timi had put the wrong antibiotic into the incubations last night. He came back later in the day to reinoculate, but this time with Kan antibiotic!

EDIT THIS: So the gels I ran today for the digestions didn't turn out. At least no band is less than 1000 bp. Our Cp-Lacpi parts should either be 90 bp for CP(x) - LacPi1 [might be inherently difficult to see] and 235 bp for CP(x) - LacPi2, for reference. Just to do a little troubleshooting, is it possible that the digestions could have failed? Maybe you accidentally enabled heated lid? I'll try to catch Mordacq after my 3 p.m. class to see what he has to say. The files are cplacpiscreengel1(9262010) and cplacpiscreengel2(9262010), cplacpiscreengel2overexposed(9262010) if you want to take a look.

Also, the bands in the gel were a little wavy. According to Mordacq, this can happen when the agarose is not properly melted all the way. I usually microwave for 1:30. I'll let it go for 30 seconds, then quickly swish it around, and put it back for the remaining 1:00 so it boils all the way. This is more important when not using low melting point agarose.

Finally, I innoculated the CP-LacPi-GFP parts again today around 7:00. I'll pop them in the centrifuge after sys phys to let them grow for a good amount of time and leave them in there for when you get in lab at 2:00.

I'll also prepare the two parts for sequencing from the last gel and give them to Mordacq at 3:00.

Also, here's what we need to get done in the near future...

- make alkaline miniprep lysis, resuspension and neutralization buffer

 --> need to ask Mordacq where glucose is and how to prepare 1% SDS

- research plate reader protocols for plate reader exps - fill out project notebook with what we've been working on

9/27/10

Inserted LacP-RBS into Tet plasmid --> transformed into ChiA knockouts

Kevin went in between classes to spin down the incubations from last night.

Timi picked up from there and miniprepped the samples and then digested them.

9/28/10

pMAL - realized primers are bad, reordering

Timi went in early to do 20 uL digests of the CP-LacPI assemblies. BUT she forgot to put in restriction enzymes. FAIL.

EDIT: Still, Kevin wanted to send you the results of the gels.

Unfortunately, even with digesting more DNA, it is still too difficult to visualize the small inserts. This can be several things (1) We just don't have any positive inserts -- which is a possibility given several unexplainable bands above 1000 bp. (2) We don't have enough DNA in the gels to properly visualize it. (3) Something with the gels/apparatus/procedure itself.

As far as controlling for DNA run on the gels, we will have to be careful loading the DNA into the gels (given the large volume, there were a few wells where I lost DNA during the transfer) as well as the volumes for our digestions (some of the digestions were not 20 µl -- this can be from liquid evaporating, but since the volumes were inconsistent it can also be pipeting error). Just some things to be aware of.

In the meantime, we should ask Mordacq what he thinks our next step should be -- whether we want to try again or try another method of screening. We should also double check we're doing the screening process correctly by digesting and running the CP-LACPI-GFP assemblies you just miniprepped. It will be much easier to see the inserts on a gel.

I'm going to send some things in for sequencing tomorrow morning so I may try to digest before class or from 11 a.m. - 1 p.m. Would you be able to run and image a gel tomorrow later afternoon/evening? Not sure if you have Jumpstart, so just let me know.

9/29/10

Site-directed mutagenesis: Amplification and DpnI digestion completed.

Ragan had some trouble with the TBE buffer. We are looking into what went wrong with it. Sean?

9/30/10

Transformation for Site-Directed mutagenesis completed.

At the lab meeting this morning, Timi discovered that Kevin had been looking for an incorrectly-sized band in the gels. They went back through the photos of gels later in the day, but were inconclusive. They are going to back-track a little bit to figure out if any of the colonies that they had picked were actually positive.

10/1/10

The transformation resulted in a good number of colonies for site-directed mutagenesis. 10 liquid cultures were made.

Timi went in early to pour a big and small gel. BUT she forgot to put in the combs. FAIL.

Kevin repoured the gels and ran the digested DNA on it. However, he forgot to run the digested parts on it as well, as the advisors had suggested.

10/2/10

Timi and Kevin put in 15 overnight inculations of 31-GFP, 32-GFP and 21-GFP.

10/3/10

Timi miniprepped the CP-LacPI-GFP cultures and digested them with E/P. She then poured a big and a small gel.

Kevin came in and ran the digested CP-LacPI-GFP, digested CP-LacPI with digested Cp and digested LacPI parts.

10/4/10

Timi and Kevin decided to take a day off :)

10/5/10

Timi and Kevin came in to discuss why their gels were running strangely. We believe there is something wrong with our buffer, so we are swapping everything out with a fresh batch of Prof. Mordacq's 0.5x TBE buffer. Timi poured a large gel that we will be using tomorrow.

10/6/10

10/7/10

Timi- took part 2-17F out of the DNA kit.

10/8/10

10/9/10

Timi- Miniprepped the 2-17F part. Digested the DNA with E/P and ligated the 2-17F GFP part into Chlor and Tet backbone.

10/10/10

Timi- Transformed and plated part 2-17F onto Chlor and Tet plates. Will be ready for miniprepping tomorrow.

10/11/10

10/12/10

10/13/10

10/14/10

10/15/10

10/16/10