Construction

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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

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

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

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

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

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

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

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

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

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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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