Team:Lethbridge/Notebook/Lab Work/June

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Contents

June 2010

June 1/2010

JV quantified the amount of DNA in gels run to date using ImageJ software. Results to be posted in working plasmids box.

Objective: Transform plasmids into DH5α
Method: Follow competent cell transformation protocol to transform the following:
From our ligations:

  • pLacI-sRBS-Lumazine-dT
  • pLacI-sRBS-Lumazine-dT
  • mms6 (A6)
  • mms6 (B6)
  • xylE (C4)
  • xylE (B4)

From the 2010 Parts Distribution:

  • ECFP (Bba_E0020)
  • EYFP (Bba_E0030)
  • BglII Endonuclease (Bba_K112106)

June 2/2010

(In Lab: JV)

Objective: Isolate plasmid DNA of RBS-xylE (BBa_J33204) from DH5α cells and confirm results.

Method: "Mini-prep" the plasmid DNA using boiling lysis miniprep. Then restrict the DNA once and run on a 1% agarose gel (TAE).


Restriction Reaction

IngredientVolume(µL)
MilliQ H20 Water15.75
Orange Buffer (10x)2
pDNA (rbs-xylE)2
EcoRI0.25

Unrestricted Control

IngredientVolume(µL)
MilliQ H20 Water16
Orange Buffer (10x)2
pDNA (rbs-xylE)2

DNA was restricted for 80 minutes at 37oC.

Analyzed results on a 1% agarose gel. Load order as follows:

LaneSampleVolume
Sample (µL)
Volume Loading
Dye (µL)
1Restricted RBS-xylE102
1Unestricted RBS-xylE12
11kb Ladder22

† Added 9µL MilliQ H2O
†† Added 8µL MilliQ H2O
Ran gel at 100V from 2 hours.
Results:

100602 JV rbs-xylE.JPG

Conclusions: Plasmid DNA prep and restriction was successful.

Objective: Ligate rbs-xylE (Bba_J33204) to our double terminator, and insert it into the pSB1T3 plasmid backbone.
Method:

  • Restrictions
    • Restrict rbs-xylE wit EcoRI and SpeI (Red Buffer)
    • Restrict the double terminator with XbaI and PstI (Tango Buffer)
    • Restrict pSB1T3 with EcoRI and PstI (Red Buffer)
    Set up reactions as follows:
    ComponentVolume (µL)
    MilliQ H2O15.5
    Buffer2
    pDNA2
    Enzyme0.25 + 0.25

    Set up control reaction as follows:

    • MilliQ H2O - 16µL
    • Buffer - 2µL
    • pDNA - 2µL

    Incubated reactions for 65 minutes at 37oC
    Killed enzymes by incubating reactions for 10 minutes at 65oC

  • Ligation
    Reaction set up as follows:
    • T4 DNA ligase - 0.25µL
    • rbs-xylE - 5µL
    • dT - 3µL
    • pSB1T3 - 8µL
    • 10x Ligation Buffer - 2µL
    • MilliQ H2O - 1.75µL
    Incubated reactions overnight at room temperature (total of 19.5 hours)
    Killed enzymes by incubating reactions for 10 minutes at 80oC</ul>

    June 2/2010 - Evening

    Objective: Set up new ligations of pLacI and sRBS-Lum-dT according to Tom Knight's protocol. Previous ligation had very little DNA.
    Relevant Information:

    • Want a final mass of 25ng of each pDNA in the ligation mix.
    • Final concentration of pDNA in restriction digest should be 25-50ng/µL.
    • Tom Knight's restriction reaction is 50µL, therefore there should be 1000ng pDNA in each restriction digest.
    • Identified the following plasmids in our working plasmids box:
    Common NameLocationConcentration (ng/µL)Volume/rxn (µL)
    pLacI MaxiprepA9990~1
    pLacI (B1)A6440~2
    sRBS-Lum-dT (2)A1965~1
    sRBS-Lum-dT (1)A21145~1
    sRBS-Lum-dT MaxiprepB84780~.2
    sRBS-Lum-dTB74375~.25
    sRBS-Lum-dT (1)G2335~3
    sRBS-Lum-dT (2)G3965~2
    • Make a 1:10 dilution of sRBS-Lum-dt maxiprep (D8) and sRBS-Lum-dT (B7). 0.5µL pDNA in 4.5µL water.
    • Cut pLacI with EcoRI and SpeI
    • Cut sRBS-Lum-dT with XbaI and PstI
    • Cut pSB1T3 with EcoRI and PstI
    • Will have total of 12 ligation reactions, want 12x2µL of pSB1T3 to add to each, therefore want 25µL of pSB1T3.

    Method:
    Restriction

    Name[pDNA] (ng/µL)Volume
    pDNA (µL)
    Volume
    Water (µL)
    Volume
    Buffer (µL)
    EnzymesTotal Volume
    sRBS-Lum-dT (A1)965143.550.25µL XbaI
    0.25µL PstI
    50
    sRBS-Lum-dT (A2)1145143.550.25µL XbaI
    0.25µL PstI
    50
    pLacI Maxiprep (A1)990143.550.25µL EcoRI
    0.25µL SpeI
    50
    sRBS-Lum-dT Maxiprep(B8)47802 (of 1:10 dilution)42.550.25µL XbaI
    0.25µL PstI
    50
    sRBS-Lum-dT (B7)43752.5 (of 1:10 dilution)4250.25µL XbaI
    0.25µL PstI
    50
    pLacI (D6)440242.550.25µL EcoRI
    0.25µL SpeI
    50
    sRBS-Lum-dT (G2)335341.550.25µL XbaI
    0.25µL PstI
    50
    sRBS-Lum-dT (G3)540242.550.25µL XbaI
    0.25µL PstI
    50
    pSB1T32512.5750.25µL EcoRI
    0.25µL PstI
    50

    Incubate for 30 minutes at 37oC (Start- 12:10pm; End- 12:40pm)
    Heat kill enzymes at 80oC for 20 minutes

    Ligation:
    In a 10µL final volume, add:

    • 2µL of sRBS-Lum-dT component
    • 2µL of pLacI component
    • 2µL of pSB1T3 component
    • 1µL of T4 Buffer
    • 0.25µL of T4 DNA Ligase
    • 2.75µL of MilliQ H2O

    Incubate for 30 minutes at room temperature to ligate
    Incubate for 20 minutes at 80oC to heat kill

    June 3/2010

    Carried out protocol described in June 2/2010 - Evening
    Analyzed results on 1% agarose gel.Load order as follows:

    LaneGel 1
    Sample
    Gel 1 LoadGel 2
    Sample
    Gel 2 Load
    11kb Ladder2µL dye, 2µL ladder
    8µL MilliQ H2O
    1kb Ladder2µL dye, 2µL ladder
    8µL MilliQ H2O
    2Restricted
    sRBS-Lum-dT (A1)
    10µL DNA
    2µL Dye
    pSB1T3 Ligation of:
    pLacI(A9)+sRBS-Lum-dT(A1)
    10µL DNA
    2µL Dye
    3Unrestricted
    sRBS-Lum-dT (A1)
    10µL DNA
    2µL Dye
    pSB1T3 Ligation of:
    pLacI(D6)+sRBS-Lum-dT(A1)
    10µL DNA
    2µL Dye
    4Restricted
    sRBS-Lum-dT (A2)
    10µL DNA
    2µL Dye
    pSB1T3 Ligation of:
    pLacI(A9)+sRBS-Lum-dT(A2)
    10µL DNA
    2µL Dye
    5Unrestricted
    sRBS-Lum-dT (A2)
    10µL DNA
    2µL Dye
    pSB1T3 Ligation of:
    pLacI(D6)+sRBS-Lum-dT(A2)
    10µL DNA
    2µL Dye
    6Restricted
    sRBS-Lum-dT (B8)
    10µL DNA
    2µL Dye
    pSB1T3 Ligation of:
    pLacI(A9)+sRBS-Lum-dT(B7)
    10µL DNA
    2µL Dye
    7Unrestricted
    sRBS-Lum-dT (B8)
    10µL DNA
    2µL Dye
    pSB1T3 Ligation of:
    pLacI(D6)+sRBS-Lum-dT(B7)
    10µL DNA
    2µL Dye
    8Restricted
    sRBS-Lum-dT (B7)
    10µL DNA
    2µL Dye
    pSB1T3 Ligation of:
    pLacI(A9)+sRBS-Lum-dT(G2)
    10µL DNA
    2µL Dye
    9Unrestricted
    sRBS-Lum-dT (B7)
    10µL DNA
    2µL Dye
    pSB1T3 Ligation of:
    pLacI(D6)+sRBS-Lum-dT(G2)
    10µL DNA
    2µL Dye
    10Restricted
    sRBS-Lum-dT (G2)
    10µL DNA
    2µL Dye
    pSB1T3 Ligation of:
    pLacI(D6)+sRBS-Lum-dT(G3)
    10µL DNA
    2µL Dye
    11Unrestricted
    sRBS-Lum-dT (G2)
    10µL DNA
    2µL Dye
    pSB1T3 Ligation of:
    pLacI(A9)+sRBS-Lum-dT(G3)
    10µL DNA
    2µL Dye
    12Restricted
    sRBS-Lum-dT (G3)
    10µL DNA
    2µL Dye
    pSB1T3 Ligation of:
    pLacI(D6)+sRBS-Lum-dT(B8)
    10µL DNA
    2µL Dye
    13Unrestricted
    sRBS-Lum-dT (G3)
    10µL DNA
    2µL Dye
    pSB1T3 Ligation of:
    pLacI(A9)+sRBS-Lum-dT(B8)
    10µL DNA
    2µL Dye
    14Restricted
    pLacI (A9)
    10µL DNA
    2µL Dye
    Restricted
    rbs-xylE
    10µL DNA
    2µL Dye
    15Unrestricted
    pLacI (A9)
    10µL DNA
    2µL Dye
    Unrestricted
    rbs-xylE
    10µL DNA
    2µL Dye
    16Restricted
    pLacI B1 (D6)
    10µL DNA
    2µL Dye
    Restricted
    pSB1T3
    10µL DNA
    2µL Dye
    17Unrestricted
    pLacI B1 (D6)
    10µL DNA
    2µL Dye
    Unrestricted pSB1T3 10µL DNA
    2µL Dye
    18Unrestricted
    dT
    10µL DNA
    2µL Dye
    pSB1T3 Ligation of:
    rbs-xylE+dT
    10µL DNA
    2µL Dye
    19Restricted
    dT
    10µL DNA
    2µL Dye

    Ran gel at 100V for 90 minutes.
    Results:

    100603JV.jpg

    Conclusions:


    June 3/2010 - Evening

    Objective: Repeat restriction of pSB1T3 and ligate with pLacI and sRBS-Lum-dT. Previous ligations all used up on gel.
    Method:
    Ligation:
    In a 10µL final volume, add:

    • 2µL of sRBS-Lum-dT component
    • 2µL of pLacI component
    • 2µL of pSB1T3 component
    • 1µL of T4 Buffer
    • 0.25µL of T4 DNA Ligase
    • 2.75µL of MilliQ H2O

    Incubate for 30 minutes at room temperature to ligate
    Incubate for 20 minutes at 80oC to heat kill
    Following ligation, transformed using transformation protocol. Plates incubated in 37oC incubator for 44 hours.
    Results: Only plate pLacI (D6) + sRBS-Lum-dT (G2) + pSB1T3 grew; had 2 colonies. Control plate did not grow, acidentally plated on tetracycline plate instead of ampicillin (pUC19).
    Follow-up: Inoculated 5mL LB media (tetracycline positive) with cells from the transformation plates and incubated at 37oC overnight. (June 5/2010).

    Objective: Ligate pBad-TetR part with fluorescent protein part in pSB1C3 backbone.
    Method:
    Restriction

    NameVolume
    pDNA (µL)
    Volume
    Water (µL)
    Volume
    Buffer (µL)
    EnzymesTotal Volume
    pSB-NEYFP (B4).843.750.25µL XbaI
    0.25µL PstI
    50
    pSB-CEYFP (B5).943.650.25µL XbaI
    0.25µL PstI
    50
    pBad-TetR).344.250.25µL EcoRI
    0.25µL SpeI
    50
    NEYFP (E1)4.340.750.25µL XbaI
    0.25µL PstI
    50
    NEYFP (E2)0.342.250.25µL XbaI
    0.25µL PstI
    50
    Fusion CEYFP (E3)3.940.650.25µL XbaI
    0.25µL PstI
    50
    Fusion CEYFP (E4)2.042.550.25µL XbaI
    0.25µL PstI
    50
    Fusion CEYFP (E5)3.041.550.25µL XbaI
    0.25µL PstI
    50
    CEYFP (E6)0.643.950.25µL XbaI
    0.25µL PstI
    50
    CEYFP (E7)0.544.050.25µL XbaI
    0.25µL PstI
    50
    pBad-TetR (F4)2.54250.25µL EcoRI
    0.25µL SpeI
    50
    pBad-TetR (F5)1.742.850.25µL EcoRI
    0.25µL SpeI
    50
    pSB-CEYFP (G4)2.941.650.25µL XbaI
    0.25µL PstI
    50
    pSB1C315.5460.25µL EcoRI
    0.25µL PstI
    62

    Incubated at 37oC for 75 minutes.

    • Used Red buffer for the EcoRI/SpeI and EcoRI/PstI digests
    • Used Tango buffer for the XbaI/PstI digests
    • Did not heat kill upon removal from incubation, put directly into -20oC fridge.

    Continue Ligation on Saturday (See below).

    June 5/2010

    (In the lab:AS)
    Objective: Ligate restriction products from June 3/2010.
    Relevant information:

    • Have 3 tubes of part 1 (pBad-TetR)
      • In ampicillin backbone
    • Have 10 tubes of part 2 (fluorescent protein - various)
      • In ampicillin backbone
    • Will have 30 combinations
    • Will use pSB1C3 as plasmid backbone
      • Used most of the pSB1T3 and want to save remainder for creating new backbone via PCR.

    Method:
    *Restriction digests were not heat killed after reactions. Freezing probably killed the restriction enzymes, but I will hea kill them at 80oC for 20 minutes anyways prior to adding Ligase.

      • Cool on ice for 10 minutes before adding ligase.
    Master MixVolume/tube (µL)Total Volume (µL)
    DNA6---
    10x Buffer132
    T4 DNA Ligase.258
    MilliQ H2O2.7588
    • Add 4µL master mix to each DNA tube.

    Follow-up: Ligation reactions will be transformed into DH5α cells


    June 6/2010

    (In Lab: JV, HS)

    Objective:
    Isolate the following plasmid DNA from DH5α:

    • pLacI-sRBS-Lumazine-dT in pSB1T3 (colony 2)
    • pLacI-sRBS-Lumazine-dT in pSB1T3 (colony 1)

    Method:
    Followed boiling lysis miniprep protocol. Eluted with 10µL Milli-Q H2O and RNase A.

    Notes:

    • Placed colony 2 in cell E10 of glycerol stocks and J6 of working plasmid box.
    • Placed colony 2 in cell F1 of glycerol stocks and J5 of working plasmid box.

    Objective:
    Transformed the following plasmid DNA into DH5α cells:

    • pBad-TetR-CEYFP: (F5+E6), (B10+E7), (B10+E6), (F4+E6), (F5+E4), (F4+E7)
    • pBad-TetR-Fusion CEYFP: (F5+E3), (B10+E4), (F4+E3), (B10+E3), (B10+E5), (F4+E4), (F5+E4), (F5+E5), (F4+E5)
    • pBad-TetR-pSB CEYFP: (F4+B5), (B10+G4), (F4+G4), (F4+B5), (F5+B5), (F5+G4)
    • pBad-TetR-NEYFP: (F5+E1), (B10+E1), (F4+E2), (F5+E2), (B10+E2), (F4+E1)
    • pBad-TetR-pSB NEYFP: (F5+B4), (F4+B4), (B10+B4)
    • Positive control -> DH5α + pSB1C3
    • Negative control -> DH%α + Milli-Q H2O

    Method:
    Followed Competent Cell Transformation protocol and used chloramphenicol as an antibiotic. We plated all 200µL of DNA onto the plates. The plates were incubated at 37oC from 4:30pm to 10:00am.

    Results:
    None of the plates showed any growth.

    June 8/2010

    (In the lab: JV)
    Objective: Follow the overexpression of our pLacI-sRBS-Lum-dT construct.
    Method: FILL ME OUT!!!!!! Results:

    TimeOD600 F1OD600 E10
    00.1180.103
    300.1330.111
    300.1450.116
    90
    1200.1600.124
    1500.1200.093
    1800.1290.100
    2100.1450.122
    2400.1580.145
    2700.1710.178
    3000.1940.222
    3300.2230.280
    3600.2520.364
    3900.2960.458
    4200.3380.557
    4500.3940.656
    4800.4530.675
    5100.5300.688
    5400.5980.706
    6000.6330.752
    6600.653
    7200.679
    1.278

    † Overexpression induced by adding 1mM IPTG.
    Following overexpression, 1mL of cells was removed from the culture, spun down at ~13000xg for 20 seconds, excess media removed and rinsed with water.
    Suspended cells in 8M urea, mixed with 6x dye and ran on 18% SDS-PAGE gel for 90 minutes at 200V. Gel stained overnight.

    Results: IMAGE TO COME!!!!