Construction

From 2006.igem.org

Revision as of 16:43, 30 September 2006

September 29, 2006

Charles:

• Prepared o/n of competent cells (DH5a-z1 and DH5a), and UT3 ABCD to miniprep again

To-Do List:

• Make new competent DH5a-z1
• Prepare o/n of regular DH5a
• Plated P0 (2006) using new competent cells
• Double digest UT1 (host) with SpeI/PstI in Buffer 2 and double digest P0352/P0456 (2005) with XbaI/PstI in Buffer 3

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September 28, 2006

Andy:

• Miniprep P0456 (2005)
• Checked the lengths (EcoRI and PstI, Buffer EcoRI): UT3 ABCDE, P0456 (2005) and P0352 (2005)
• Transformed P0152, P0352, P0452, and P0456 (2006). [Plated on Amp plates, because no more Amp/Kan plate]

To-Do List:

• Plate DH5a cells
• Make LB Broth, plates and print protocols
• Digest P0352 and P0456 (2005) and ligate with UT1
• Prepare o/n of UT3 ABCDE again and try to miniprep again

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September 27, 2006

Charles:

• Made freezer stocks of UT2 x 2 and UT3 x 2

Andy, Natalie:

• Mini-preppred UT3 B(AB) C(BB) D(BC) E(CC)
• Transformed and plated P0452 (2005) (On Amp rather than proper AK)
• Checked lengths of UT3 BCDE (inconclusive due to anomalous bands, D appears promising - yellow rating), P0152 (2nd check, Extra band, wrong insert length), P0352 (2nd check, not the best bands, correct lengths - changed rating from red to yellow)
• Prepared o/n of P0456 (2005) for Miniprep
• Added part lengths and resistance info in spreadsheet

To-Do List:

• Miniprep P0456 (2005)
• Take P0456, P0452, P0152, P0352 from 2006 registry
• Check lengths of UT3 BCDE and P0352 again
• Make more plates, make more LB
• Learn to use new DH5a (with no LacI expression) for testing
• Post up new protocols

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September 26, 2006

Charles:

• Prepared o/n of UT2 (2 vials) and UT3 (2 vials) for a freezer stock

Andy:

• Prepared o/n of UT3 BCDE for mini-preps
• transformed and plated P0452 (2005) and P0456 (2005)

To-Do List:

• Make freezer stock and minipreps of above tubes and check lengths along with P0352 (2005) and P0152 (2005)
• Make more plates, autoclave tips, make more LB
• Post up new protocols, include part length and resistance info in spreadsheet

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September 22, 2006

To-Do List:

• Make frozen stock of UT2 and UT3
• Prepare o/n for UT3 BCDE for mini-prep
• Post up new protocols, include part length and resistance info in spreadsheet
• Make more plates and re-plate P0452 (2005) – AK, and P0456 (2005) – AC

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September 21, 2006

Charles:

• Made 22 competent cells
• Tested Module 1 using varying concentrations of IPTG (0, 2, 20, 200, 2000 uM) to induce GFP (E0240 (2005)) and mRFP (I13507 (2005))
• Data indicates that the GFP and mRFP as well as the LacI+ pL promoter (R0011) are functional (Does not indicate functionality of the pBad/AraC promoter and the inverter)
• Some of the replicates seem to be outliers and if excluded, the regression increases significantly
• DH5a-z1 are known to fluoresce, so the fact that untransformed cells glow brighter is not a big concern
• Next step is to transform UT2 and UT3 into LacI negative cells then:
  • Characterize the fluorescence under varying Arabinose concentrations.
  • Vary the temperature to verify the temperature sensitivity of LacI ts and its affects on reporter expression

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  To-Do List:

  • Post up new protocols, include part length and resistance info in spreadsheet
  • Make more plates and re-plate P0452 (2005) – AK, and P0456 (2005) – AC

  [http://2006.igem.org/University_of_Toronto_2006 Home]

  September 20, 2006

  Natalie, Andy:

  • Prepared o/n growth of UT3 BCDE for mini-prep, DH5a for competent cells, and UT2 – 3,4,5, UT3 – 7,8,9 and DH5a x 2 (control) for making fluorescence measurements.
  • Checked lengths of miniprep UT2 ABCDEF, UT3 A (possibly unreliable due to 2 day o/n growth), P0352 (2005), and P0152 (2005).

  To-Do List:

  • Make 24 competent cells
  • Miniprep UT3 BCDE and test lengths
  • Make more plates and re-plate P0452 (2005) – AK, and P0456 (2005) – AC
  • Do measurements on UT2 and UT3
  • Post up new protocols, include part length and resistance info in spreadsheet

  [http://2006.igem.org/University_of_Toronto_2006 Home]

  September 18, 2006

  Charles:

  • Only mini-prepped UT3 A because they were grown for two o/n (Just want to see what happens)
  • Prepared new o/n for UT3 BCDE as well as 1 DH5a-z1, 2 UT2 and 2 UT3 for testing tomorrow. And 1 DH5a-z1 for making competent cells

  To-Do List:

  • Make 24 competent cells
  • Test the lengths of UT2 ABCEDF, P0352 (2005), P0152 (2005), and UT3 BCDE
  • Make more plates and re-plate P0452 (2005) – AK, and P0456 (2005) – AC
  • Prepare new o/n for UT3 BCDE – for mini-prep
  • Prepare o/n in the following quantities: 3 DH5a-z1, 3 UT2 and 3 UT3

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  September 16, 2006

  Charles:

  • Made 4 replications of UT2 in 200 uM, 100 uM, 50 uM, 25 uM and 0 uM of IPTG.
    
    
    
    
    • OD of cells is too high, need to be <= 1.2
    • Need a control of untransformed DH5a-z1 to compare the relative fluorescence
  • Made frozen stock (2 vials each): UT01, I13507 (2005), B0015 (2005), B0031 (2005)
  • Mini-prepped UT2 ABCEDF, P0352 (2005) and P0152 (2005)

  To-Do List:

  • Test the lengths of UT2 ABCEDF, P0352 (2005) and P0152 (2005)
    • Prepare an o/n growth of the “best” UT2 for testing
    • Prepare an o/n growth of regular DH5a-z1
  • Mini-prep UT03 ABCD and test their lengths along with P00352 (2005) and P0152 (2005)
  • Re-plate P0452 (2005) - AK, and P0456 (2005) - AC
  • Make 24 competent cells

  [http://2006.igem.org/University_of_Toronto_2006 Home]

  September 15, 2006

  Andy, Charles, Natalie, Stan, Elliott:

  • Checked and transformed I+J+I plasmids (Bands: ~6000-5000, 3500-2500 (smear))
  • Made competent cells (although, may have to repeat)
  • Prepared o/n growths of I+J+E (12 vials), P0152 (2005) and P0352 (2005). P0456 (2005) and P0452 (2005) did not seem to grow very well so left them in the incubator for a 2nd night
  • Made 8 Amp plates

  To-Do List:

  • Mini-prep P0152 (2005), P0352 (2005) and 6 of the I+J+E
  • Try to get J04450 (2006) working again.
  • Check lengths of R0011 (2005/6), if no good, then obtain R0010 (2005/6) from Registry
  • Make freezer stock (2 each) of I+J, B0015 (2005), B0031 (2005), and I13507 (2005) and put in -80C freezer stock
  • Test the fluorescence of I+J+E

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