Construction

From 2006.igem.org

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Contents 1 October 14, 2006 2 October 13, 2006 3 October 12, 2006 4 October 11, 2006 5 October 8, 2006 6 October 7, 2006 7 October 6, 2006 8 October 4, 2006

October 14, 2006

Charles, Jovan:

• Mini-prepped UT2 2/4 and UT3 7
• Transform UT2 2/4 and UT3 7 into DH5a-z1 and DH5a cells
• Continue with the IPTG test for the rest of the colonies that we weren’t able to test yesterday

To-Do List:

• Measure absorbance spectrum of Arabinose
• Make o/n of working UT2/UT3 for repeat of IPTG test.
• Look into using tetR and tet pL instead of cI and Prm+

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October 13, 2006

Charles, Jovan, Nick:

• Prepared tubes at 1:20 dilution of UT2/UT3 o/n for fluorescence at 2000 uM IPTG
• Prepared tubes at 1:20 dilution of DH5a o/n for LacI repression at 0%, 0.02%, 0.2% and 2% Arabinose
• Digested I12006 ABCD (2005), I12006 AB (2006) with EcoRI/SpeI (which was a mistake)
• Checked UT2/UT3 and we found the original glowing colonies (UT2 1/2 and UT3 7).
  • UT2 (2) Plate BB, colony 21 and (4) Plate CA, colony 23
  • UT3 (7) Plate BB, colony 26
  • Couldn’t take pictures due time constraints

To-Do List:

• Verify last day’s results through fluorescence microscopy and mini-prep and transform DH5a-z1 and DH5a
• Wait for Natalie to bring the biobricks from Waterloo to obtain I12006 and J04450

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October 12, 2006

Andy, Charles, Melinda, Natalie, Ram, Siva, Tara:

• Made new agar plates (amp = 5, kan = 5, amp/kan = 5)
• Made o/n of UT2 (5), UT3 (5) and DH5a
  • UT2:
    1. Plate BA, colony 20
    2. Plate BB, colony 21
    3. Plate CA, colony 22
    4. Plate CA, colony 23
    5. Plate CB, colony 24
  • UT3:
    1. Plate AB, colony 25
    2. Plate BB, colony 26
    3. Plate BB, colony 27
    4. Plate BC, colony 28
    5. Plate CC, colony 29
  • Also made o/n for UT2 (2), UT3 (2) from freezer stock
• Transformed I12006 AB (2005)
• Digested and checked lengths
  • I12006 ABH (2005): (5000 – 4000) – correct
  • UT1 AB: (5000 – 4000, 3500 – 3000) – correct
  • UT2 EF (3000 – 2500, 1000 – 750) – not so correct
• Digested I12006 AB (2005) with SpeI/PstI and E0240 EF (2005) with XbaI/PstI
  • I12006 ABH (2005): (5000 – 4000, 2000 – 1500) – not so correct
  • E0240 EF (2005): (3500 – 3000, 2500 – 2000, 2000 – 1500, 1000 – 750) – not correct
  • Did not gel extract I12006 and E0240

To-Do List:

• Try different ligation enzymes, thus ligate UT4 with I12006 then ligate with E0240.
  • Digest I12006 AB (2005) with EcoRI/XbaI (host) and UT4 AB with EcoRI/SpeI (insert)
• Check UT2/UT3 under microscope to find out if we have fluorescence
  • If we do, mini-prep the o/n and transform cells
• DH5a vs. [Arabinose] to test potential arabinose induced fluorescence reduction
  • Use PBS + Arabinose as a reference

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October 11, 2006

To-Do List:

• Digest I12006 H (2005) and I12006 AB (2006) with XbaI/SpeI and check lengths
• Transform cells with I12006 AB (2005)
• Transform cells with UT1 AB, UT2 EF, UT4 AB
  • Try to find the UT4 used to make a measurement (that was successful)
• Digest I12006 AB (2005) with SpeI/PstI
  • Ligate I12006 AB (2005) and E0240 (purified DNA from Oct4)

Long-Term Goals:

• Once the mini-preps of the correct lengths are obtained, retest UT2/UT3 with increasing IPTG to repeat previous experiment (if successful, take pictures)
• Need to test to see if arabinose is having deleterious effects on fluorescence
  • DH5a auto-fluorescence vs. [Arabinose]
  • Consider using a tetR promoter instead of pBad/araC
• Do an Arabinose and IPTG surface response test to find optimal concentrations
• NOTE: try not to overgrow the cells – signal seems to saturate quickly

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October 8, 2006

Anne:

• Prepared DH5a UT2 for LacI temperature test
• Mini-prepped I2006 H (2005) and I12006 AB (2006)

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October 7, 2006

Cheng:

• Prepared DH5a-z1 UT2 for IPTG induction of GFP

Conrad:

• Re-test UT4C, UT5 ABC and I12006 EFG (2005) with XbaI/SpeI
• Miniprep UT2/3 in DH5a

Siva:

• Prepared DH5a UT2 for Arabinose induced LacI test

Recorded digest length check results:

• I12006 EFG (4000-5000, 1500-1000)
• UT5 ABC (3500-4000, 750-1000)
• UT4 C (3000-3500)
• UT3 DH5a (750-1000, 2000-2500)
• UT2 DH5a (750-1000, 2000-2500)

Test Results

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October 6, 2006

Andy, HoKwon:

• Prepared o/n (3 vials each) of DH5a and DH5a-z1 with UT2 and UT3 for testing
• Prepared o/n of UT2 DH5a and UT3 DH5a for miniprep
• Transformed and plated I12006 (2005) (poured in amp plate first by accident), I12006 (2006)
• Double Digest parts E0240 CD (2005), UT4 AB with XbaI/SpeI
  • E0240 CD (2000-2500, 750-1000) Correct!
  • UT4 AB (3000-4000) Correct!

Charles, Cheng, Nick:

• Re-tested UT4 C, UT5 ABC – failed due to a very dry gel

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October 4, 2006

Andy:

• Checked lengths (EcoRI, PstI) of following
  • I12006 EFG (4000-5000, 1000-1500 for all three)
  • UT4 AB (3000-4000, 2000) C (3000-4000, 250)
  • UT5 ABC (2500-3000, 750-1000)
• Digested following
  • I12006 (2005) E (6000 by SpeI and PstI)
  • E0240 (2005) C (2500-3000, 2000-2500, 1500-2000, 750-1000 by XbaI and PstI)
  • UT4 AB (6000-10000, 2000-2500 by SpeI and PstI) C (5000-6000, 3000 by SpeI and PstI)
  • UT5 ABC (3000-4000 by SpeI and PstI)
• Gel extracted E0240 (insert) and UT4 AB (host)
• Transformed and plated DH5a with UT2/UT3 for testing
• Made o/n of UT2/UT3 with IPTG

To-Do:

• Redo transformation of I12006 (2005)
• Learn to take fluorescent images under microscope
• Temperature testing
• Try ligating UT5 again
• Running out of water and PCR tubes

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