(Difference between revisions)
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Redid PCR of CHS3 for pMAL insert
Redid PCR of CHS3 for pMAL insert
TODO: pour plates, characterize ligation in terms of procedure variability
TODO: make more parts and make glycerol stock of parts

Revision as of 15:32, 3 August 2010

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6/14-18/10 (Boot Camp)

DNA extracted from kit

Part: [1]


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


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)


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.


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.


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).


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


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.


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).


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).


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).


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)


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)


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).


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)


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


Finished the ethanol precipiation (RESULTS)

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


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


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)


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.


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.


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


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.


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.


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.


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.


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.


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.


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.


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


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


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


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


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


Competency test (iGEM T10 from Leonard T10) in progress

CHS3 pMAL insertion Assembly in progress ((nde1)-CHS3-(EcoR1) in pMAL)

CHS3 test-vector Assembly in progress (lacp-RBS-CHS3-2XT in cpA/K)

CP/Lacpi test-vector Assembly in progress (2x3) (CP-Lacpi-GFP)

Holin test-vector Assembly in progress (either CP-Lacpi-RBS-Holin-2XT or Lacp-RBS-Holin-2XT)

cP 3A Assembly troubleshooting: white colonies? <- Running 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

TODO: Glycerol Stock of parts in 4 degree




(CP-Lacpi) pick multiple colonies, purify, digest with E/P and gel -> should be 35+1372/1370 = 1405/1407 +20(pre/suffix)

(Holin-2XT) pick multiple colonies, purify, digest with E/P and gel -> should be 1257, 317 + 129 = 1386, 446 +20 (prefix/suffix) (holin 1, holin 2)