Construction

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<center>
<center>
-
<font size=4>< [[Aug11-Sep7]] | [[Sep8-14]] ></font>
+
<font size=4>< [[Aug11-Sep7]] | [[Sep8-14]] | [[Sep15-26]] | [[Sep27-Oct3]] | [[Oct4-Oct13]] | [[Construction]] ></font>
</center>
</center>
-
== September 27, 2006 ==
+
== October 30, 2006 ==
'''Charles:'''
'''Charles:'''
<ul>
<ul>
-
     <li>Made freezer stocks of UT2 x 2 and UT3 x 2
+
    <li>We sent in our parts to MIT!
 +
    <li>Re mini-prepped UT9 ABCDE and digested using XbaI/PstI
 +
    <li>UT9 ABCDE – (2500 – 2000, 1000 – 750) – Correct!
 +
    <li>Gel extracted and quantitated: got no bands =(
 +
     <li>Made Freezer stock of DH5a and DH5a-z1
 +
    <li>Made o/n of UT14 and pBAD for mini-prep, DH5a and DH5a-z1 for competent cells
 +
    <li>Tested Module 2 with constant 0.2% arabinose and increasing aTc from 0, 5, 10, 20, 50 ng/mL for both UT10 and DH5a
 +
    <li>Made o/n of UT2/UT3 and UT10/UT11 for possible testing, UT9 BCDEFG for miniprep and gel extract
</ul>
</ul>
-
'''Andy, Natalie:'''
+
'''To-Do List:'''
<ul>
<ul>
-
     <li>Mini-preppred UT3 B(AB) C(BB) D(BC) E(CC)
+
    <li>Make more M9 media
-
     <li>Transformed and plated P0452 (2005) (On Amp rather than proper AK)
+
    <li>Make competent cells
-
     <li>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)
+
    <li>Ligate final parts together
-
     <li>Prepared o/n of P0456 (2005) for Miniprep
+
</ul>
-
     <li>Added part lengths and resistance info in spreadsheet
+
 
 +
== October 29, 2006 ==
 +
 
 +
'''Charles:'''
 +
<ul>
 +
    <li>Gel extracted:
 +
    <ul>
 +
        <li>UT10 A = 14 ng/uL
 +
        <li>UT10 B = 16 ng/uL
 +
        <li>UT10 C = 14 ng/uL
 +
        <li>UT11 ABC ~ 0 
 +
    </ul>
 +
    <li>Made o/n of DH5a and DH5a-z1, as well as UT10/UT11 ABC for aTc testing
 +
     <li>Mini-prepped UT9 DE, UT14 AB, pBAD 33 AB
 +
    <li>Length check: (none worked)
 +
     <ul>
 +
        <li>UT9 DE (S/P), UT14 AB (E/S), pBAD A (S/P) -- none worked
 +
    </ul>
 +
     <li>made more o/n of UT9 ABCDE and UT10/UT11 DE
 +
</ul>
 +
 
 +
== October 28, 2006 ==
 +
 
 +
'''Charles:'''
 +
<ul>
 +
    <li>Mini-prepped UT9, UT10, UT11 ABC (may be slightly messed up – miniprep again?)
 +
    <li>Length check:
 +
    <ul>
 +
        <li>I0500 – no bands =(
 +
        <li>Q04400 (2005/2006) – correct
 +
        <li>Q03400 - correct
 +
        <li>UT9 – no bands =( try again tomorrow with concentrated DNA
 +
        <li>UT10 - correct
 +
        <li>UT11 - correct
 +
    </ul>
 +
    <li>UT9 looks red
 +
     <li>Tested UT10/11 under +aTc = 50 ng/mL and –aTc = 0 ng/mL conditions
 +
    <li>Plated freezer stock of DH5a and DH5a-z1 to make a new plate
 +
     <li>Made o/n of pBAD AB and UT9 DEF for miniprep
</ul>
</ul>
'''To-Do List:'''
'''To-Do List:'''
 +
- Make o/n of DH5a and DH5a-z1
 +
- Miniprep pBAD AB
 +
- Make Amp and Kan plates
 +
 +
== October 27, 2006 ==
 +
 +
'''Charles, Nick:'''
<ul>
<ul>
-
     <li>Miniprep P0456 (2005)
+
     <li>M9 Test – 0% - 2% Arabinose in UT3 and DH5a (control), temperature test at 0.2% Arabinose,
-
     <li>Take P0456, P0452, P0152, P0352 from 2006 registry
+
        IPTG verification at 0% and 2% arabinose:
-
    <li>Check lengths of UT3 BCDE and P0352 again
+
     <ul>
-
    <li>Make more plates, make more LB
+
        <li>Dilute o/n of UT3 and DH5a in 1:50 o/n to LB ratio in appropriate arabinose/IPTG
-
     <li>Learn to use new DH5a (with no LacI expression) for testing
+
            concentrations – incubate 3 hrs
-
     <li>Post up new protocols
+
        <li>Resuspend in 50:50 M9:LB (including arabinose/IPTG) and divided 0.2% arabinose for
 +
            temperature test – incubate for 3 hrs
 +
        <li>Resuspend in M9 media – incubate for 5 hrs
 +
        <ul>
 +
            <li>First 3 hrs without signalling conditions, then latter 2 hrs with conditions
 +
        </ul>
 +
        <li>Read with fluorometer (PBS wash not required)
 +
    </ul>
 +
     <li>Ligated I0500 B (2005) + I13507 (2005) / I13504 (2005/2006)
 +
     <li>Made o/n of UT9, UT10, UT11 ABC
 +
    <li>Made o/n of DH5a and DH5a-z1
</ul>
</ul>
-
[http://2006.igem.org/University_of_Toronto_2006 Home]
+
'''To-Do List:'''
 +
<ul>
 +
    <li>Check lengths of I0500 (2006), Q04400 (2005/2006), Q03400 (2006)
 +
    <li>Miniprep and check lengths of UT9, UT10, UT11 – if correct, wait for functionality test before transforming into cells
 +
    <li>Make competent cells (DH5a and DH5a-z1)
 +
    <li>Make Amp and Kan plates
 +
</ul>
-
== September 26, 2006 ==
+
== October 26, 2006 ==
-
'''Charles:'''
+
'''Andy, Natalie, Charles, Tara, HoKwon:'''
<ul>
<ul>
-
     <li>Prepared o/n of UT2 (2 vials) and UT3 (2 vials) for a freezer stock
+
     <li>Ligated and transformed UT6, UT7 with I13504 (2005/2006) (Name UT10, UT11) (Quantitation showed correct band for all)
 +
    <li>Ligated and transformed R0011 (2005) with I13507 (2005) (Name UT9) (Quantitation showed correct band for all)
 +
    <li>Made o/n of UT3 x 3, DH5a for testing
 +
    <li>Quantitated I0500 (2005) AB to be ligated to I13504 (2005/2006) (Band: 6000 - roughly correct. A 20 ng/uL B 300 ng/uL)
</ul>
</ul>
 +
 +
'''To-do list:'''
 +
<ul>
 +
    <li>Make o/n of UT9, UT10, UT11 for miniprep
 +
    <li>Ligate I0500 (2005) with I13504 (2005/2006)
 +
    <li>M9 testing of UT3
 +
</ul>
 +
 +
== October 25, 2006 ==
'''Andy:'''
'''Andy:'''
<ul>
<ul>
-
     <li>Prepared o/n of UT3 BCDE for mini-preps
+
     <li>Minipreppred UT6, UT7, UT8
-
    <li>transformed and plated P0452 (2005) and P0456 (2005)  
+
    <li>Gel-extracted (only for correct bands)
 +
          <ul>         
 +
              <li>UT6 S/P (band: 8000-10000 correct) -- 80 ng/uL
 +
              <li>UT7 S/P (band: 8000-10000 correct) -- 50 ng/uL
 +
              <li>UT8 S/P (band: 6000 incorrect)
 +
              <li>I13504 (2005) X/P (band: 750-1000, 1500-2000 correct) -- 140 ng/uL
 +
              <li>I13504 (2006) X/P (band: 750-1000, 1500-2000 correct) -- 90 ng/uL
 +
          </ul>
 +
    <li>Made o/n of UT2 x 3, UT3 x 3, DH5a for testing
</ul>
</ul>
-
'''To-Do List:'''
+
'''To-do list:'''
<ul>
<ul>
-
     <li>Make freezer stock and minipreps of above tubes and check lengths along with P0352 (2005) and P0152 (2005)
+
     <li>Quantitate and ligate UT6, UT7 with I13504 (2005/2006)
-
     <li>Make more plates, autoclave tips, make more LB
+
     <li>Quantitate and ligate R0011 (2005) with I13507 (2005)
-
     <li>Post up new protocols, include part length and resistance info in spreadsheet
+
     <li>M9 testing of UT2, UT3
</ul>
</ul>
-
[http://2006.igem.org/University_of_Toronto_2006 Home]
+
== October 24, 2006 ==
-
== September 22, 2006 ==
+
'''Melinda:'''
 +
<ul>
 +
    <li>Minipreppred and length-checked (unsuccessful) I13522 (2006), I13504 (2005/2006) AB, R0011 (2005)
 +
    <li>Made o/n of UT6-8 for miniprep
 +
</ul>
-
'''To-Do List:'''
+
== October 23, 2006 ==
 +
 
 +
'''Andy:'''
<ul>
<ul>
-
     <li>Make frozen stock of UT2 and UT3
+
     <li>Gel-extrated I13507 (2005) X/P
-
     <li>Prepare o/n for UT3 BCDE for mini-prep
+
     <li>Made o/n of I13522 (2006), I13504 (2005/2006) x 2, R0011 (2005) for minipreps
-
     <li>Post up new protocols, include part length and resistance info in spreadsheet
+
     <li>Tested UT3 for temperature and arabinose sensitivity, plated UT2, UT3, DH5a for plate test
-
     <li>Make more plates and re-plate P0452 (2005) – AK, and P0456 (2005) – AC
+
     <li>Made plates
 +
    <li>ligated and transformed UT1 with Q4400 (2005) = UT6, Q3400 (2006) = UT7, and Q4400 (2006) = UT8
</ul>
</ul>
-
[http://2006.igem.org/University_of_Toronto_2006 Home]
+
'''To-do List:'''
 +
<ul>
 +
    <li>Quantitate and Ligate R0011 (2005) with I13507 (2005)
 +
    <li>Miniprep and length-check I13522 (2006), I13504 (2005/2006) x 2, R0011 (2005)
 +
    <li>Make multiple o/n of UT6-8 for miniprep
 +
</ul>
-
== September 21, 2006 ==
+
== October 22, 2006 ==
-
'''Charles:'''
+
'''Massive To-do List'''
 +
 
 +
The following is a guideline.  Day-to-day details need to be filled in as we progress.  Change colors from red to black as each task is completed.
 +
 
 +
'''Construction:'''
 +
<ul>
 +
    <li>Transform and miniprep I13522 (2006) (amp) from registry DNA-2 15H
 +
    <li>Transform and miniprep I13504 (2005/2006) (amp) from registry DNA-1 12D (2005/2006)
 +
    <li><font color=red>Miniprep</font> 2 UT1 for ligation (not urgent)
 +
    <li>Ligate UT1 with Q03400, Q04400 (2005/2006) (4 ligations) (Name UT6-9)
 +
    <li>If possible, <font color=red>ligate</font> R0011 (2005) with I13507 (2005) in parallel (Name UT10)
 +
    <li><font color=red>Ligate</font> UT6-9 with I13504 (4 ligations) (Name UT11-14)
 +
    <li><font color=red>Ligate</font> UT11-14 with UT10 (4 ligations) (Nam UT15-18)
 +
</ul>
 +
 
 +
'''Testing:'''
 +
<ul>
 +
    <li>Temperature-test and Arabinose/IPTG-test UT3 (See below for protocol)
 +
    <li>Plate-test UT2, UT3, DH5a, UT13, I13522 (See below for protocol)
 +
</ul>
 +
 
 +
'''Andy:'''
<ul>
<ul>
-
     <li>Made 22 competent cells
+
     <li>Gel-extrated R0011 (2005) S/P (band: 2000 - correct)
-
     <li>Tested Module 1 using varying concentrations of IPTG (0, 2, 20, 200, 2000 uM) to induce GFP
+
     <li>Cannot gel-extract I13507 (2005) X/P because there was no more extraction column (bands: 2500-3000, 2000-2500, 750-1000 - correct)
-
        (E0240 (2005)) and mRFP (I13507 (2005))
+
     <li>Transformed R0011 (2005), I13522 (2006), I13504 (2005/2006)
-
    <table>
+
     <li>Quantitated
-
        <tr><td colspan=2>[[Image:Table_GFP.jpg]]</td>
+
-
        <tr><td>[[Image:Graph1_GFP.jpg]]</td><td>[[Image:Graph2_GFP.jpg]]</td>
+
-
        <tr><td>[[Image:Table_mRFP.jpg]]</td><td>[[Image:Graph_mRFP.jpg]]</td>
+
-
    </table>
+
-
     <li>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)
+
-
     <li>Some of the replicates seem to be outliers and if excluded, the regression increases
+
-
        significantly
+
-
    <li>DH5a-z1 are known to fluoresce, so the fact that untransformed cells glow brighter is not a
+
-
        big concern
+
-
    <li>Next step is to transform UT2 and UT3 into LacI negative cells then:
+
         <ul>
         <ul>
-
            <li>Characterize the fluorescence under varying Arabinose concentrations.
+
        <li>Q3400 (2005) E/X (no band)
-
            <li>Vary the temperature to verify the temperature sensitivity of LacI ts and its affects
+
        <li>Q4400 (2005) E/X (band: 5000 - correct 120 ng/uL)
-
                on reporter expression
+
        <li>Q3400 (2006) E/X (band: 5000 - correct 24 ng/uL)
 +
        <li>Q4400 (2006) E/X (band: 5000 - correct 120 ng/uL)
 +
        <li>UT1 D E/S (band: 2500-3000 - correct 18 ng/uL)
 +
        <li>UT1 E E/S (band: 2500-3000 - correct 12 ng/uL)
 +
        <li>UT1 F E/S (band: 2500-3000 - correct 30 ng/uL)
         </ul>
         </ul>
 +
    <li>o/n of UT3 x 5, UT2 x 1, and DH5a x 4 for temperature, arabinose, and plate testing
 +
</ul>
 +
 +
'''To-do List:'''
 +
<ul>
 +
    <li>Ligate UT1 with Q3400 (2006), Q4400 (2005/2006)
 +
    <li>Make o/n of I13522 (2006) for miniprep and plate-test, I13504 (2005/2006), I13507 (2005) for miniprep
 +
    <li>Gel-extract I13507 (2005) X/P
 +
    <li>Temperature, arabinose, and plate testing
 +
    <li>Purchase gel-extraction kit, make Amp plates
 +
</ul>
 +
 +
'''Guidelines for Plate-test:'''
 +
<ol>
 +
    <li>On an Amp plate, make 4 sectors (500 uL?) with varying arabinose (0.02%, 0.2%, 2%, 20%) and making sure they don’t touch each other (otherwise diffusion will happen and they’ll be the same concentration), let the arabinose area dry.
 +
    <li>Take o/n of UT3-DH5a and take out 200 uL and add 800 uL of fresh LB and incubate for 1 hr.
 +
    <li>Spin down the cells at 3000 RPM for 5 min and remove 800 uL of supernatant.  Resuspend cells with the remaining 200 uL and gently spread onto plate
 +
</ol>
 +
 +
'''Guidelines for Temperature-test:'''
 +
<ol>
 +
    <li>Put a constant amount of arabinose (either 0.02% or 0.2% - don’t want TOO much repression) in each dilution.
 +
    <li>Grow dilutions at 37C until the absorbance is ~0.6 using the GFP ABS program.  Then take some tubes out and put 1 mL into an Eppendorf tube.
 +
    <li>Use the white filter caps and put them on each Eppendorf tube (to allow air in, but to protect against contamination)
 +
    <li>Set mixer to the equivalent to the shaker in the incubator (remember it’s a smaller tube, so there probably needs to be more force on it to get it to shake).
 +
    <li>Set temperature to 22C
 +
    <li>Check the absorbance after a couple of hours and prepare the samples when the absorbance matches the absorbance of the tubs in the incubator (these should be ready before the 22C ones are ready)
 +
    <li>Hopefully we’ll see a significant difference in fluorescence: cold should fluoresce less than hot.
 +
</ol>
 +
 +
 +
[http://2006.igem.org/University_of_Toronto_2006 Home]
 +
 +
== October 19, 2006 ==
 +
 +
'''Melinda:'''
 +
<ul>
 +
    <li>Miniprepped Q04400 (2005/2006) and Q04510 (2005/2006)
 +
    <li>Made o/n of Q03400 (2005/2006), (3) UT2-DH5a, (2) DH5a, and (1) DH5a/DH5a-z1 for freezer stock and fresh plate
</ul>
</ul>
'''To-Do List:'''
'''To-Do List:'''
<ul>
<ul>
-
     <li>Post up new protocols, include part length and resistance info in spreadsheet
+
     <li>Miniprep Q03400 (2005/2006)
-
     <li>Make more plates and re-plate P0452 (2005) – AK, and P0456 (2005) – AC
+
    <li>Repeat Oct 18 test, except with GFP and proper controls
 +
     <li>Digest and check J04450 (W), Q03400, Q04400, and Q04510 (2005/2006).  If correct, move on with gel extraction and ligation.
 +
    <li>Make 20% Arabinose, and 2 blank agar plates
</ul>
</ul>
[http://2006.igem.org/University_of_Toronto_2006 Home]
[http://2006.igem.org/University_of_Toronto_2006 Home]
-
== September 20, 2006 ==
+
== October 18, 2006 ==
-
'''Natalie, Andy:'''
+
'''Andy, Konstantin:'''
<ul>
<ul>
-
     <li>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.
+
     <li>Transformed and plated Q03400 (2005) (2006)
-
     <li>Checked lengths of miniprep UT2 ABCDEF, UT3 A (possibly unreliable due to 2 day o/n growth), P0352 (2005), and P0152 (2005).
+
    <li>Gel-extracted UT1 DEF with EcoRI/SpeI (2500-3000, 5000, 8000), 8000 band bright, digestion appeared incomplete
 +
    <li>Made A, K, AK plates
 +
     <li>Made o/n of Q04400 (2005) (2006) Q04510 (2005) (2006)
 +
    <li>Tested UT3 in DH5a with 0%-2% arabinose
</ul>
</ul>
'''To-Do List:'''
'''To-Do List:'''
<ul>
<ul>
-
     <li>Make 24 competent cells
+
     <li>Make o/n of Q03400 (2005) (2006)
-
     <li>Miniprep UT3 BCDE and test lengths
+
     <li>Miniprep Q04400 (2005) (2006) and Q04510 (2005) (2006)
-
    <li>Make more plates and re-plate P0452 (2005) – AK, and P0456 (2005) – AC
+
     <li>Replate UT1 with miniprep from a good conlony
-
     <li>Do measurements on UT2 and UT3
+
     <li>Miniprep Q04400 (2005) (2006) Q04510 (2005) (2006) and check to see if bands are correct.  If
-
     <li>Post up new protocols, include part length and resistance info in spreadsheet
+
        so, digest with EcoRI/XbaI and gel extract
 +
    <li>Ligate with UT1
</ul>
</ul>
[http://2006.igem.org/University_of_Toronto_2006 Home]
[http://2006.igem.org/University_of_Toronto_2006 Home]
-
== September 18, 2006 ==
+
== October 17, 2006 ==
-
'''Charles:'''
+
'''Melinda:'''
<ul>
<ul>
-
     <li>Only mini-prepped UT3 A because they were grown for two o/n  (Just want to see what happens)
+
     <li>Double Digetsed J04450 (W), UT1 DEFG with XbaI/SpeI using Buffer 2
-
     <li>Prepared new o/n for UT3 BCDE as well as 1 DH5a-z1, 2 UT2 and 2 UT3 for testing tomorrow. And
+
    <li>Results:
-
         1 DH5a-z1 for making competent cells
+
    <ul>
 +
        <li>J04450 (W) (1 band)
 +
        <li>UT1 D (5000 – 4000, 3000 – 2500)
 +
        <li>UT1 E (2 bands)
 +
        <li>UT1 F (2 bands)
 +
        <li>UT1 G (2 bands)
 +
    </ul>
 +
     <li>Transformed DH5a cells with Q04400 (2005/2006), Q04510 (2005/2006)
 +
    <li>Made o/n of DH5a and (3 vials) of DH5a UT3
 +
</ul>
 +
 
 +
'''Note – Change in protocol:'''
 +
<ul>
 +
    <li>After incubation for 1 hour, centrifuge tubes for 5 min @ 3000 RPM to pellet cells
 +
    <li>Remove 800 uL of supernatant
 +
    <li>Resuspend pellet with remaining 200 uL
 +
    <li>Spread on plate and wait ~15-20 min.
 +
</ul>
 +
 
 +
[http://2006.igem.org/University_of_Toronto_2006 Home]
 +
 
 +
== October 16, 2006 ==
 +
 
 +
'''Melinda:'''
 +
<ul>
 +
    <li>Checked parts, including the relevant parts from Waterloo (W):
 +
    <ul>
 +
        <li>I0500 (W): (5000 – 4000, 1500 – 1000) – correct!
 +
        <li>J04450 (W): (2500 – 2000) – plasmid is correct, but no part?
 +
        <li>J06801 (W): (8000 – 6000) – not correct!
 +
        <li>E0240 (W): (4000 – 3500, 3500 – 3000, 1000 – 750) – part is correct?
 +
        <li>I12006 (W): (5000 – 4000, 2500 – 2000)  - plasmid is correct, undigested plasmid?
 +
        <li>UT2 2: (3500 – 3000, 2500 – 2000, 2000 – 1500) – correct, extra band?
 +
         <li>UT2 4: (3500 – 3000, 2500 – 2000, 2000 – 1500) – correct, extra band?
 +
        <li>UT3 7: (3500 – 3000, 2500 – 2000) – correct!
 +
    </ul>
 +
    <li>Viewed cells induced by IPTG under microscope and verified the functionality of UT2/UT3 in DH5a and DH5a-z1
</ul>
</ul>
'''To-Do List:'''
'''To-Do List:'''
<ul>
<ul>
-
     <li>Make 24 competent cells
+
     <li>Repeat Double Digest of J04450 (W) and UT1 ABCD with XbaI/SpeI (Run undigested plasmids beside each double digest)
-
    <li>Test the lengths of UT2 ABCEDF, P0352 (2005), P0152 (2005), and UT3 BCDE
+
     <li>Make more Amp plates
-
     <li>Make more plates and re-plate P0452 (2005) – AK, and P0456 (2005) – AC
+
     <li>If digest is successful, transform DH5a cells with the successful parts
-
     <li>Prepare new o/n for UT3 BCDE – for mini-prep
+
     <li>Find tetR replacements for cI and ligate those parts together.
-
     <li>Prepare o/n in the following quantities: 3 DH5a-z1, 3 UT2 and 3 UT3
+
</ul>
</ul>
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-
== September 16, 2006 ==
+
== October 15, 2006 ==
'''Charles:'''
'''Charles:'''
<ul>
<ul>
-
     <li>Made 4 replications of UT2 in 200 uM, 100 uM, 50 uM, 25 uM and 0 uM of IPTG.
+
     <li>Made o/n of the newly plated UT2 2/4 and UT3 7 as well as UT2/UT3 freezer stock
-
    <br><br><center>[[Image:Module01_Test01_Raw.jpg]]<br>
+
-
        [[Image:Module01_Test01_Graph.jpg]]</center><br>
+
-
        <ul>
+
-
            <li>OD of cells is too high, need to be <= 1.2
+
-
            <li>Need a control of untransformed DH5a-z1 to compare the relative fluorescence
+
-
        </ul>
+
-
    <li>Made frozen stock (2 vials each): UT01, I13507 (2005), B0015 (2005), B0031 (2005)
+
-
    <li>Mini-prepped UT2 ABCEDF, P0352 (2005) and P0152 (2005)
+
</ul>
</ul>
'''To-Do List:'''
'''To-Do List:'''
<ul>
<ul>
-
     <li>Test the lengths of UT2 ABCEDF, P0352 (2005) and P0152 (2005)
+
     <li>Make sure the cells fluoresce by diluting in IPTG
-
        <ul>
+
-
            <li>Prepare an o/n growth of the “best” UT2 for testing
+
-
            <li>Prepare an o/n growth of regular DH5a-z1
+
-
        </ul>
+
-
    <li>Mini-prep UT03 ABCD and test their lengths along with P00352 (2005) and P0152 (2005)
+
-
    <li>Re-plate P0452 (2005) - AK, and P0456 (2005) - AC
+
-
    <li>Make 24 competent cells
+
</ul>
</ul>
[http://2006.igem.org/University_of_Toronto_2006 Home]
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-
== September 15, 2006 ==
+
== October 14, 2006 ==
-
'''Andy, Charles, Natalie, Stan, Elliott:'''
+
'''Charles, Jovan:'''
<ul>
<ul>
-
     <li>Checked and transformed I+J+I plasmids (Bands: ~6000-5000, 3500-2500 (smear))
+
     <li>Mini-prepped UT2 2/4 and UT3 7
-
     <li>Made competent cells (although, may have to repeat)
+
     <li>Transform UT2 2/4 and UT3 7 into DH5a-z1 and DH5a cells
-
     <li>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
+
     <li>Continue with the IPTG test for the rest of the colonies that we weren’t able to test
-
         very well so left them in the incubator for a 2nd night
+
         yesterday
-
    <li>Made 8 Amp plates
+
</ul>
</ul>
'''To-Do List:'''
'''To-Do List:'''
<ul>
<ul>
-
     <li>Mini-prep P0152 (2005), P0352 (2005) and 6 of the I+J+E
+
     <li>Measure absorbance spectrum of Arabinose
-
     <li>Try to get J04450 (2006) working again.
+
     <li>Make o/n of working UT2/UT3 for repeat of IPTG test.
-
     <li>Check lengths of R0011 (2005/6), if no good, then obtain R0010 (2005/6) from Registry
+
     <li>Look into using tetR and tet pL instead of cI and Prm+
-
    <li>Make freezer stock (2 each) of I+J, B0015 (2005), B0031 (2005), and I13507 (2005) and put in -80C freezer stock
+
-
    <li>Test the fluorescence of I+J+E
+
</ul>
</ul>
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Latest revision as of 08:14, 2 November 2006

< Aug11-Sep7 | Sep8-14 | Sep15-26 | Sep27-Oct3 | Oct4-Oct13 | Construction >

Contents

October 30, 2006

Charles:

  • We sent in our parts to MIT!
  • Re mini-prepped UT9 ABCDE and digested using XbaI/PstI
  • UT9 ABCDE – (2500 – 2000, 1000 – 750) – Correct!
  • Gel extracted and quantitated: got no bands =(
  • Made Freezer stock of DH5a and DH5a-z1
  • Made o/n of UT14 and pBAD for mini-prep, DH5a and DH5a-z1 for competent cells
  • Tested Module 2 with constant 0.2% arabinose and increasing aTc from 0, 5, 10, 20, 50 ng/mL for both UT10 and DH5a
  • Made o/n of UT2/UT3 and UT10/UT11 for possible testing, UT9 BCDEFG for miniprep and gel extract

To-Do List:

  • Make more M9 media
  • Make competent cells
  • Ligate final parts together

October 29, 2006

Charles:

  • Gel extracted:
    • UT10 A = 14 ng/uL
    • UT10 B = 16 ng/uL
    • UT10 C = 14 ng/uL
    • UT11 ABC ~ 0 
  • Made o/n of DH5a and DH5a-z1, as well as UT10/UT11 ABC for aTc testing
  • Mini-prepped UT9 DE, UT14 AB, pBAD 33 AB
  • Length check: (none worked)
    • UT9 DE (S/P), UT14 AB (E/S), pBAD A (S/P) -- none worked
  • made more o/n of UT9 ABCDE and UT10/UT11 DE

October 28, 2006

Charles:

  • Mini-prepped UT9, UT10, UT11 ABC (may be slightly messed up – miniprep again?)
  • Length check:
    • I0500 – no bands =(
    • Q04400 (2005/2006) – correct
    • Q03400 - correct
    • UT9 – no bands =( try again tomorrow with concentrated DNA
    • UT10 - correct
    • UT11 - correct
  • UT9 looks red
  • Tested UT10/11 under +aTc = 50 ng/mL and –aTc = 0 ng/mL conditions
  • Plated freezer stock of DH5a and DH5a-z1 to make a new plate
  • Made o/n of pBAD AB and UT9 DEF for miniprep

To-Do List: - Make o/n of DH5a and DH5a-z1 - Miniprep pBAD AB - Make Amp and Kan plates

October 27, 2006

Charles, Nick:

  • M9 Test – 0% - 2% Arabinose in UT3 and DH5a (control), temperature test at 0.2% Arabinose, IPTG verification at 0% and 2% arabinose:
    • Dilute o/n of UT3 and DH5a in 1:50 o/n to LB ratio in appropriate arabinose/IPTG concentrations – incubate 3 hrs
    • Resuspend in 50:50 M9:LB (including arabinose/IPTG) and divided 0.2% arabinose for temperature test – incubate for 3 hrs
    • Resuspend in M9 media – incubate for 5 hrs
      • First 3 hrs without signalling conditions, then latter 2 hrs with conditions
    • Read with fluorometer (PBS wash not required)
  • Ligated I0500 B (2005) + I13507 (2005) / I13504 (2005/2006)
  • Made o/n of UT9, UT10, UT11 ABC
  • Made o/n of DH5a and DH5a-z1

To-Do List:

  • Check lengths of I0500 (2006), Q04400 (2005/2006), Q03400 (2006)
  • Miniprep and check lengths of UT9, UT10, UT11 – if correct, wait for functionality test before transforming into cells
  • Make competent cells (DH5a and DH5a-z1)
  • Make Amp and Kan plates

October 26, 2006

Andy, Natalie, Charles, Tara, HoKwon:

  • Ligated and transformed UT6, UT7 with I13504 (2005/2006) (Name UT10, UT11) (Quantitation showed correct band for all)
  • Ligated and transformed R0011 (2005) with I13507 (2005) (Name UT9) (Quantitation showed correct band for all)
  • Made o/n of UT3 x 3, DH5a for testing
  • Quantitated I0500 (2005) AB to be ligated to I13504 (2005/2006) (Band: 6000 - roughly correct. A 20 ng/uL B 300 ng/uL)

To-do list:

  • Make o/n of UT9, UT10, UT11 for miniprep
  • Ligate I0500 (2005) with I13504 (2005/2006)
  • M9 testing of UT3

October 25, 2006

Andy:

  • Minipreppred UT6, UT7, UT8
  • Gel-extracted (only for correct bands)
    • UT6 S/P (band: 8000-10000 correct) -- 80 ng/uL
    • UT7 S/P (band: 8000-10000 correct) -- 50 ng/uL
    • UT8 S/P (band: 6000 incorrect)
    • I13504 (2005) X/P (band: 750-1000, 1500-2000 correct) -- 140 ng/uL
    • I13504 (2006) X/P (band: 750-1000, 1500-2000 correct) -- 90 ng/uL
  • Made o/n of UT2 x 3, UT3 x 3, DH5a for testing

To-do list:

  • Quantitate and ligate UT6, UT7 with I13504 (2005/2006)
  • Quantitate and ligate R0011 (2005) with I13507 (2005)
  • M9 testing of UT2, UT3

October 24, 2006

Melinda:

  • Minipreppred and length-checked (unsuccessful) I13522 (2006), I13504 (2005/2006) AB, R0011 (2005)
  • Made o/n of UT6-8 for miniprep

October 23, 2006

Andy:

  • Gel-extrated I13507 (2005) X/P
  • Made o/n of I13522 (2006), I13504 (2005/2006) x 2, R0011 (2005) for minipreps
  • Tested UT3 for temperature and arabinose sensitivity, plated UT2, UT3, DH5a for plate test
  • Made plates
  • ligated and transformed UT1 with Q4400 (2005) = UT6, Q3400 (2006) = UT7, and Q4400 (2006) = UT8

To-do List:

  • Quantitate and Ligate R0011 (2005) with I13507 (2005)
  • Miniprep and length-check I13522 (2006), I13504 (2005/2006) x 2, R0011 (2005)
  • Make multiple o/n of UT6-8 for miniprep

October 22, 2006

Massive To-do List

The following is a guideline. Day-to-day details need to be filled in as we progress. Change colors from red to black as each task is completed.

Construction:

  • Transform and miniprep I13522 (2006) (amp) from registry DNA-2 15H
  • Transform and miniprep I13504 (2005/2006) (amp) from registry DNA-1 12D (2005/2006)
  • Miniprep 2 UT1 for ligation (not urgent)
  • Ligate UT1 with Q03400, Q04400 (2005/2006) (4 ligations) (Name UT6-9)
  • If possible, ligate R0011 (2005) with I13507 (2005) in parallel (Name UT10)
  • Ligate UT6-9 with I13504 (4 ligations) (Name UT11-14)
  • Ligate UT11-14 with UT10 (4 ligations) (Nam UT15-18)

Testing:

  • Temperature-test and Arabinose/IPTG-test UT3 (See below for protocol)
  • Plate-test UT2, UT3, DH5a, UT13, I13522 (See below for protocol)

Andy:

  • Gel-extrated R0011 (2005) S/P (band: 2000 - correct)
  • Cannot gel-extract I13507 (2005) X/P because there was no more extraction column (bands: 2500-3000, 2000-2500, 750-1000 - correct)
  • Transformed R0011 (2005), I13522 (2006), I13504 (2005/2006)
  • Quantitated
    • Q3400 (2005) E/X (no band)
    • Q4400 (2005) E/X (band: 5000 - correct 120 ng/uL)
    • Q3400 (2006) E/X (band: 5000 - correct 24 ng/uL)
    • Q4400 (2006) E/X (band: 5000 - correct 120 ng/uL)
    • UT1 D E/S (band: 2500-3000 - correct 18 ng/uL)
    • UT1 E E/S (band: 2500-3000 - correct 12 ng/uL)
    • UT1 F E/S (band: 2500-3000 - correct 30 ng/uL)
  • o/n of UT3 x 5, UT2 x 1, and DH5a x 4 for temperature, arabinose, and plate testing

To-do List:

  • Ligate UT1 with Q3400 (2006), Q4400 (2005/2006)
  • Make o/n of I13522 (2006) for miniprep and plate-test, I13504 (2005/2006), I13507 (2005) for miniprep
  • Gel-extract I13507 (2005) X/P
  • Temperature, arabinose, and plate testing
  • Purchase gel-extraction kit, make Amp plates

Guidelines for Plate-test:

  1. On an Amp plate, make 4 sectors (500 uL?) with varying arabinose (0.02%, 0.2%, 2%, 20%) and making sure they don’t touch each other (otherwise diffusion will happen and they’ll be the same concentration), let the arabinose area dry.
  2. Take o/n of UT3-DH5a and take out 200 uL and add 800 uL of fresh LB and incubate for 1 hr.
  3. Spin down the cells at 3000 RPM for 5 min and remove 800 uL of supernatant. Resuspend cells with the remaining 200 uL and gently spread onto plate

Guidelines for Temperature-test:

  1. Put a constant amount of arabinose (either 0.02% or 0.2% - don’t want TOO much repression) in each dilution.
  2. Grow dilutions at 37C until the absorbance is ~0.6 using the GFP ABS program. Then take some tubes out and put 1 mL into an Eppendorf tube.
  3. Use the white filter caps and put them on each Eppendorf tube (to allow air in, but to protect against contamination)
  4. Set mixer to the equivalent to the shaker in the incubator (remember it’s a smaller tube, so there probably needs to be more force on it to get it to shake).
  5. Set temperature to 22C
  6. Check the absorbance after a couple of hours and prepare the samples when the absorbance matches the absorbance of the tubs in the incubator (these should be ready before the 22C ones are ready)
  7. Hopefully we’ll see a significant difference in fluorescence: cold should fluoresce less than hot.


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October 19, 2006

Melinda:

  • Miniprepped Q04400 (2005/2006) and Q04510 (2005/2006)
  • Made o/n of Q03400 (2005/2006), (3) UT2-DH5a, (2) DH5a, and (1) DH5a/DH5a-z1 for freezer stock and fresh plate

To-Do List:

  • Miniprep Q03400 (2005/2006)
  • Repeat Oct 18 test, except with GFP and proper controls
  • Digest and check J04450 (W), Q03400, Q04400, and Q04510 (2005/2006). If correct, move on with gel extraction and ligation.
  • Make 20% Arabinose, and 2 blank agar plates

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October 18, 2006

Andy, Konstantin:

  • Transformed and plated Q03400 (2005) (2006)
  • Gel-extracted UT1 DEF with EcoRI/SpeI (2500-3000, 5000, 8000), 8000 band bright, digestion appeared incomplete
  • Made A, K, AK plates
  • Made o/n of Q04400 (2005) (2006) Q04510 (2005) (2006)
  • Tested UT3 in DH5a with 0%-2% arabinose

To-Do List:

  • Make o/n of Q03400 (2005) (2006)
  • Miniprep Q04400 (2005) (2006) and Q04510 (2005) (2006)
  • Replate UT1 with miniprep from a good conlony
  • Miniprep Q04400 (2005) (2006) Q04510 (2005) (2006) and check to see if bands are correct. If so, digest with EcoRI/XbaI and gel extract
  • Ligate with UT1

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October 17, 2006

Melinda:

  • Double Digetsed J04450 (W), UT1 DEFG with XbaI/SpeI using Buffer 2
  • Results:
    • J04450 (W) (1 band)
    • UT1 D (5000 – 4000, 3000 – 2500)
    • UT1 E (2 bands)
    • UT1 F (2 bands)
    • UT1 G (2 bands)
  • Transformed DH5a cells with Q04400 (2005/2006), Q04510 (2005/2006)
  • Made o/n of DH5a and (3 vials) of DH5a UT3

Note – Change in protocol:

  • After incubation for 1 hour, centrifuge tubes for 5 min @ 3000 RPM to pellet cells
  • Remove 800 uL of supernatant
  • Resuspend pellet with remaining 200 uL
  • Spread on plate and wait ~15-20 min.

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October 16, 2006

Melinda:

  • Checked parts, including the relevant parts from Waterloo (W):
    • I0500 (W): (5000 – 4000, 1500 – 1000) – correct!
    • J04450 (W): (2500 – 2000) – plasmid is correct, but no part?
    • J06801 (W): (8000 – 6000) – not correct!
    • E0240 (W): (4000 – 3500, 3500 – 3000, 1000 – 750) – part is correct?
    • I12006 (W): (5000 – 4000, 2500 – 2000) - plasmid is correct, undigested plasmid?
    • UT2 2: (3500 – 3000, 2500 – 2000, 2000 – 1500) – correct, extra band?
    • UT2 4: (3500 – 3000, 2500 – 2000, 2000 – 1500) – correct, extra band?
    • UT3 7: (3500 – 3000, 2500 – 2000) – correct!
  • Viewed cells induced by IPTG under microscope and verified the functionality of UT2/UT3 in DH5a and DH5a-z1

To-Do List:

  • Repeat Double Digest of J04450 (W) and UT1 ABCD with XbaI/SpeI (Run undigested plasmids beside each double digest)
  • Make more Amp plates
  • If digest is successful, transform DH5a cells with the successful parts
  • Find tetR replacements for cI and ligate those parts together.

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October 15, 2006

Charles:

  • Made o/n of the newly plated UT2 2/4 and UT3 7 as well as UT2/UT3 freezer stock

To-Do List:

  • Make sure the cells fluoresce by diluting in IPTG

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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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