Construction

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<font color="blue" size=4>Page [[1]] [[2]] [http://2006.igem.org/3 3]</font>
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<font size=4>< [[Aug11-Sep7]] | [[Sep8-14]] | [[Sep15-26]] | [[Sep27-Oct3]] | [[Oct4-Oct13]] | [[Construction]] ></font>
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== September 16, 2006 ==
+
== October 30, 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>We sent in our parts to MIT!
-
 
+
    <li>Re mini-prepped UT9 ABCDE and digested using XbaI/PstI
-
     <li>Made frozen stock (2 vials each): UT01, I13507 (2005), B0015 (2005), B0031 (2005)
+
    <li>UT9 ABCDE – (2500 – 2000, 1000 – 750) – Correct!
-
     <li>Mini-prepped UT2 ABCEDF, P0352 (2005) and P0152 (2005)
+
    <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>
'''To-Do List:'''
'''To-Do List:'''
<ul>
<ul>
-
     <li>Test the lengths of UT2 ABCEDF, P0352 (2005) and P0152 (2005)
+
     <li>Make more M9 media
-
        <ul>
+
     <li>Make competent cells
-
            <li>Prepare an o/n growth of the “best” UT2 for testing
+
     <li>Ligate final parts together
-
            <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>
-
== September 15, 2006 ==
+
== October 29, 2006 ==
-
'''Andy, Charles, Natalie, Stan, Elliott:'''
+
'''Charles:'''
<ul>
<ul>
-
     <li>Checked and transformed I+J+I plasmids (Bands: ~6000-5000, 3500-2500 (smear))
+
     <li>Gel extracted:
-
     <li>Made competent cells (although, may have to repeat)
+
    <ul>
-
     <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>UT10 A = 14 ng/uL
-
        very well so left them in the incubator for a 2nd night
+
        <li>UT10 B = 16 ng/uL
-
     <li>Made 8 Amp plates
+
        <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>
</ul>
-
'''To-Do List:'''
+
== October 28, 2006 ==
 +
 
 +
'''Charles:'''
<ul>
<ul>
-
     <li>Mini-prep P0152 (2005), P0352 (2005) and 6 of the I+J+E
+
     <li>Mini-prepped UT9, UT10, UT11 ABC (may be slightly messed up – miniprep again?)
-
     <li>Try to get J04450 (2006) working again.
+
     <li>Length check:
-
     <li>Check lengths of R0011 (2005/6), if no good, then obtain R0010 (2005/6) from Regsitry
+
     <ul>
-
     <li>Make freezer stock (2 each) of I+J, B0015 (2005), B0031 (2005), and I13507 (2005) and put in -80C freezer stock
+
        <li>I0500 – no bands =(
-
     <li>Test the fluorescence of I+J+E
+
        <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>
-
== September 14, 2006 ==
+
'''To-Do List:'''
 +
- Make o/n of DH5a and DH5a-z1
 +
- Miniprep pBAD AB
 +
- Make Amp and Kan plates
-
'''Andy, Natalie:'''
+
== October 27, 2006 ==
-
<table border=1 cellspacing=1>
+
'''Charles, Nick:'''
-
     <tr><th>Part Name<th>Parts Match<th>Plasmid Match
+
<ul>
-
    <tr><td>I0500 E (2005)</td><td>--</td><td>O
+
     <li>M9 Test – 0% - 2% Arabinose in UT3 and DH5a (control), temperature test at 0.2% Arabinose,
-
    <tr><td>I0500 F (2005)</td><td>--</td><td>O
+
        IPTG verification at 0% and 2% arabinose:
-
     <tr><td>I+J+E AA-CB (2005)</td><td>O</td><td>O
+
    <ul>
-
</table>
+
        <li>Dilute o/n of UT3 and DH5a in 1:50 o/n to LB ratio in appropriate arabinose/IPTG
 +
            concentrations – incubate 3 hrs
 +
        <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>
 +
'''To-Do List:'''
<ul>
<ul>
-
     <li>all the I+J+E bands were roughly in the right location (base pair bracket) ~60% confidence
+
     <li>Check lengths of I0500 (2006), Q04400 (2005/2006), Q03400 (2006)  
-
     <li>ligated I+J with I13507 (made 6 replicates)
+
     <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>
</ul>
-
'''Andy, Charles:'''
+
== October 26, 2006 ==
 +
 
 +
'''Andy, Natalie, Charles, Tara, HoKwon:'''
<ul>
<ul>
-
     <li>Made o/n of cells to be made competent
+
     <li>Ligated and transformed UT6, UT7 with I13504 (2005/2006) (Name UT10, UT11) (Quantitation showed correct band for all)
-
     <li>Transformed I+J+E into 6 plates, P0456 (2005), P0152 (2005), P0352 (2005), and P0452 (2005).
+
     <li>Ligated and transformed R0011 (2005) with I13507 (2005) (Name UT9) (Quantitation showed correct band for all)
-
     <li>Made plates: 10 Amp, 1 Amp/Cam, 3 Amp/Kan
+
     <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:'''
+
'''To-do list:'''
<ul>
<ul>
-
     <li>Make o/n growth of P0456 (2005), P0152 (2005), P0352 (2005), and P0452 (2005).
+
     <li>Make o/n of UT9, UT10, UT11 for miniprep
-
     <li>If I+J+E colonies formed, make o/n growths at the 2 extreme temperatures and 1 at 37C
+
    <li>Ligate I0500 (2005) with I13504 (2005/2006)
-
    <li>Transform/check I+J+I plasmids.
+
     <li>M9 testing of UT3
-
    <li>Make 6 Amp plates
+
-
    <li>Make 30 competent cells
+
-
    <li>Make o/n growth of I+J, B0015 (2005), B0031 (2005), and I13507 (2005) to make -80C freezer
+
-
        stock
+
</ul>
</ul>
-
== September 13, 2006 ==
+
== October 25, 2006 ==
-
Digested I13507 (2005) and extracted plasmid.  B = 15 ng/uL, C = 30 uL (had an extra band at 1500…so ignored it and hopefully we got the right part at ~3000)
+
'''Andy:'''
 +
<ul>
 +
    <li>Minipreppred UT6, UT7, UT8
 +
    <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>
-
Ligate I+J ABC with E0240 AB (2005) in all the combinations: AA, AB, BA, BB, CA, CB (part – plasmid)
+
'''To-do list:'''
-
 
+
-
To-Do List:
+
<ul>
<ul>
-
     <li>Redo digestion/ligation/quantitation page
+
     <li>Quantitate and ligate UT6, UT7 with I13504 (2005/2006)
-
    <li>Make more plates: Amp = 7, Amp/Kan = 2, Amp/Cyc = 1
+
     <li>Quantitate and ligate R0011 (2005) with I13507 (2005)
-
     <li>get following parts from the 2005 Registry: P0456 (2005) – 16 O, pSB1AC3; P0152 (2005) – 19
+
     <li>M9 testing of UT2, UT3
-
        I, pSB1A2; P0352 (2005) – 21 E, pSB1AK3; P0452 (2005) – 23 A, pSB1AK3 and transform cells
+
-
     <li>Transform cells with I+J+E (all 6)
+
</ul>
</ul>
-
== September 12, 2006 ==
+
== October 24, 2006 ==
-
Andy, Anne, Charles, Natalie:
+
'''Melinda:'''
<ul>
<ul>
-
     <li>Mini-prepped I+J HIJK
+
     <li>Minipreppred and length-checked (unsuccessful) I13522 (2006), I13504 (2005/2006) AB, R0011 (2005)
-
    <li>Checked ABCDEFG but only ABCD worked, b/c EFG had thee bands – try again later?
+
     <li>Made o/n of UT6-8 for miniprep
-
     <li>Quantitated I+J: A = 25 ng/uL; B = 15 ng/uL; C =  20 ng/uL
+
</ul>
</ul>
-
Andy, Elliott, Ting:
+
== October 23, 2006 ==
 +
 
 +
'''Andy:'''
<ul>
<ul>
-
     <li>Mini-prepped B0015 (2006) x 2, I0500 (2005) + J06801 (2006) 1:2 x 1, I0500 (2005) + J06801
+
     <li>Gel-extrated I13507 (2005) X/P
-
        (2006) 1:3 x 2.
+
    <li>Made o/n of I13522 (2006), I13504 (2005/2006) x 2, R0011 (2005) for minipreps
-
     <li>One I0500 (2005) + J06801 (2006) 1:2 overnight growth had no cells.
+
    <li>Tested UT3 for temperature and arabinose sensitivity, plated UT2, UT3, DH5a for plate test
 +
     <li>Made plates
 +
    <li>ligated and transformed UT1 with Q4400 (2005) = UT6, Q3400 (2006) = UT7, and Q4400 (2006) = UT8
</ul>
</ul>
-
To-Do List:
+
'''To-do List:'''
<ul>
<ul>
-
     <li>Make more plates: Amp = 7, Amp/Kan = 2, Amp/Cyc = 1
+
     <li>Quantitate and Ligate R0011 (2005) with I13507 (2005)
-
    <li>cut I13507 (2005) with EcoRI and XbaI with Buffer 2
+
     <li>Miniprep and length-check I13522 (2006), I13504 (2005/2006) x 2, R0011 (2005)
-
     <li>get following parts from the 2005 Registry: P0456 (2005) – 16 O, pSB1AC3; P0152 (2005) – 19   
+
     <li>Make multiple o/n of UT6-8 for miniprep
-
        I, pSB1A2; P0352 (2005) – 21 E, pSB1AK3; P0452 (2005) – 23 A, pSB1AK3 and transform cells
+
-
     <li>ligate I+J with E0240 (2005) at a 1:3 = plasmid : insert molar ratio. And double digest to
+
-
        check the lengths, meanwhile, plate the cells.
+
</ul>
</ul>
-
== September 11, 2006 ==
+
== October 22, 2006 ==
-
Made Mini-preps of B0015 (2005) – need to test tomorrow – and made o/n of B0015 (2005) for a freezer stock.  B0015 (2006) didn’t seem to grow very well so let it grow for another day and mini-prep tomorrow.
+
'''Massive To-do List'''
-
Made many (at least 6) o/n growths of the new part I0500 + J06801
+
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.
-
To-Do List:
+
'''Construction:'''
<ul>
<ul>
-
     <li>mini-prep B0015 (2006) – if cells have grown
+
     <li>Transform and miniprep I13522 (2006) (amp) from registry DNA-2 15H
-
     <li>mini-prep all the I0500 + J06801 vials.  Label them I+J
+
     <li>Transform and miniprep I13504 (2005/2006) (amp) from registry DNA-1 12D (2005/2006)
-
     <li>test the mini-preps for correct lengths (I+J will have a plasmid length of pSB2K3 and a part
+
     <li><font color=red>Miniprep</font> 2 UT1 for ligation (not urgent)
-
        length of 1210 + 1371 = 2581 bp long)
+
    <li>Ligate UT1 with Q03400, Q04400 (2005/2006) (4 ligations) (Name UT6-9)
-
     <li>if test is good, proceed to ligation with E0240 (2005) and I13507 (2005).  – NOTE: need to
+
     <li>If possible, <font color=red>ligate</font> R0011 (2005) with I13507 (2005) in parallel (Name UT10)
-
        digest the plasmid (E0240 and I13507) with EcoRI and XbaI and to digest the insert (I+J) with
+
    <li><font color=red>Ligate</font> UT6-9 with I13504 (4 ligations) (Name UT11-14)
-
        EcoRI and SpeI – this is a pre-insertion.
+
     <li><font color=red>Ligate</font> UT11-14 with UT10 (4 ligations) (Nam UT15-18)
-
     <li>Once ligation is complete, do an initial check to see if ligation was successful, if so,
+
-
        transform cells with the new parts I+J+E and I+J+I.
+
</ul>
</ul>
-
== September 10, 2006 ==
+
'''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>
-
Miniprepped a lot of parts:
+
'''Andy:'''
-
<table border=1 cellspacing=1>
+
<ul>
-
     <tr><th>Part Name<th>Parts Match<th>Plasmid Match
+
     <li>Gel-extrated R0011 (2005) S/P (band: 2000 - correct)
-
    <tr><td>I0500 C (2005)</td><td>X</td><td>O
+
     <li>Cannot gel-extract I13507 (2005) X/P because there was no more extraction column (bands: 2500-3000, 2000-2500, 750-1000 - correct)
-
     <tr><td>I0500 D (2005)</td><td>X</td><td>O
+
     <li>Transformed R0011 (2005), I13522 (2006), I13504 (2005/2006)
-
    <tr><td>E0240 C (2005)</td><td>O</td><td>O
+
     <li>Quantitated
-
     <tr><td>E0240 D (2005)</td><td>O</td><td>O
+
        <ul>
-
    <tr><td>B0031 C (2005)</td><td>--</td><td>O
+
        <li>Q3400 (2005) E/X (no band)
-
    <tr><td>B0031 B (2005)</td><td>--</td><td>O
+
        <li>Q4400 (2005) E/X (band: 5000 - correct 120 ng/uL)
-
     <tr><td>I12006 D (2005)</td><td>X</td><td>X
+
        <li>Q3400 (2006) E/X (band: 5000 - correct 24 ng/uL)
-
    <tr><td>I12006 C (2005)</td><td>X</td><td>X
+
        <li>Q4400 (2006) E/X (band: 5000 - correct 120 ng/uL)
-
    <tr><td>I13507 B (2005)</td><td>O</td><td>O
+
        <li>UT1 D E/S (band: 2500-3000 - correct 18 ng/uL)
-
    <tr><td>I13507 C (2005)</td><td>O</td><td>O
+
        <li>UT1 E E/S (band: 2500-3000 - correct 12 ng/uL)
-
    <tr><td>J06801 D (2006)</td><td>O</td><td>O
+
        <li>UT1 F E/S (band: 2500-3000 - correct 30 ng/uL)
-
    <tr><td>J06801 C (2006)</td><td>O</td><td>O
+
        </ul>
-
    <tr><td>C0056 B (2005)</td><td>X</td><td>O
+
    <li>o/n of UT3 x 5, UT2 x 1, and DH5a x 4 for temperature, arabinose, and plate testing
-
    <tr><td>C0056 C (2005)</td><td>X</td><td>O
+
</ul>
-
    <tr><td>B0034 B (2005)</td><td>--</td><td>O
+
-
    <tr><td>B0034 C (2005)</td><td>--</td><td>O
+
-
</table>
+
-
 
+
-
Made an o/n growth of B0015 (2005/2006) to have miniprepped and to make freezer stock
+
-
 
+
-
LOOKS LIKE LIGATION WORKED!!! The initial check showed that there was a band at ~4500 (plasmid), a band at ~2000 (new part) and a band at ~1000 (old part).  So, cells were transformed with the new part and plated.  It seems like 1:2 molar ratio of plasmid to insert worked the best (brightest new part and faintest old part)
+
-
To-Do List:
+
'''To-do List:'''
<ul>
<ul>
-
     <li>miniprep B0015 (2005/6) and I0500 (2005)
+
     <li>Ligate UT1 with Q3400 (2006), Q4400 (2005/2006)
-
     <li>prepare o/n growth of new part
+
     <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>
</ul>
-
== September 9, 2006 ==
+
'''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>
-
Mini-prepped everything (from yesterday)
+
'''Guidelines for Temperature-test:'''
-
Plated B0015 (2005) and B0015 (2006)
+
<ol>
-
Decided to have a larger stock of mini-preps so made double amounts of o/n growth of I0500 (2005), E0240 (2005), B0031 (2005), I12006 (2005), I13507 (2006), J06801 (2006), C0056 (2005), B0034 (2005)
+
    <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>
-
== September 8, 2006 ==
 
-
Double digested parts B0015 (2005), C0056 (2005) and I13507 (2005) again with EcoRI and PstI with Buffer EcoRI.  To ligate the parts, I0500 (2005) will be the host and J06801 (2006) will be the insert. The new part will then be then inserted into the E0240 (2005) host. Therefore, I0500 will be cut with SpeI/PstI, J06801 will be cut with XbaI/PstI, and E0240 will be cut with EcoRI/XbaI.
+
[http://2006.igem.org/University_of_Toronto_2006 Home]
-
<table border=1 cellspacing=1>
+
== October 19, 2006 ==
-
    <tr><th>Part Name<th>Parts Match<th>Plasmid Match
+
-
    <tr><td>B0015 A (2005)</td><td>O</td><td>O
+
-
    <tr><td>C0056 A (2005)</td><td>X</td><td>O
+
-
    <tr><td>I13507 A (2005)</td><td>O</td><td>O
+
-
</table>
+
-
Check c0056 A (2005) again
+
'''Melinda:'''
-
 
+
-
The quantitation results are as follows:
+
<ul>
<ul>
-
     <li>E0240 B, 8.0 ng/uL
+
     <li>Miniprepped Q04400 (2005/2006) and Q04510 (2005/2006)
-
     <li>E0240 A, 6.0 ng/uL
+
     <li>Made o/n of Q03400 (2005/2006), (3) UT2-DH5a, (2) DH5a, and (1) DH5a/DH5a-z1 for freezer stock and fresh plate
-
    <li>I0500 B, 13.5 ng/uL
+
</ul>
</ul>
-
To-Do List:
+
'''To-Do List:'''
<ul>
<ul>
-
     <li>transform cells with B0015 (2005)
+
     <li>Miniprep Q03400 (2005/2006)
-
     <li>MP B0031 (2005), B0034 (2005), C0056 (2005), E0240 (2005), I0500 (2005), I12006 (2005),
+
     <li>Repeat Oct 18 test, except with GFP and proper controls
-
        I13507 (2005), J06801 (2006)
+
    <li>Digest and check J04450 (W), Q03400, Q04400, and Q04510 (2005/2006).  If correct, move on with gel extraction and ligation.
-
     <li>Ligate I0500 + J06801
+
     <li>Make 20% Arabinose, and 2 blank agar plates
</ul>
</ul>
-
== September 7, 2006 ==
+
[http://2006.igem.org/University_of_Toronto_2006 Home]
-
Obtained purified DNA of I0500 (2005) with a concentration of 0 ng/uL.  Try again tomorrow
+
== October 18, 2006 ==
-
Made mini-preps of B0015 (2005) and I13507 (2005) and made a plate of B0031 (2005)
+
'''Andy, Konstantin:'''
-
 
+
<ul>
-
<table border=1 cellspacing=1>
+
     <li>Transformed and plated Q03400 (2005) (2006)
-
     <tr><th>Part Name<th>Part Description<th>Parts Match<th>Plasmid Match
+
    <li>Gel-extracted UT1 DEF with EcoRI/SpeI (2500-3000, 5000, 8000), 8000 band bright, digestion appeared incomplete
-
    <tr><td>B0015 (2005)</td><td>Terminator</td><td>--</td><td>--
+
     <li>Made A, K, AK plates
-
     <tr><td>B0031 (2005)</td><td>Medium RBS</td><td>--</td><td>O
+
    <li>Made o/n of Q04400 (2005) (2006) Q04510 (2005) (2006)
-
    <tr><td>C0056 (2005)</td><td>Repressor protein 434 cI</td><td>--</td><td>O
+
     <li>Tested UT3 in DH5a with 0%-2% arabinose
-
     <tr><td>I13507 (2005)</td><td>RBS.mRFP.Terminator</td><td>--</td><td>--
+
</ul>
-
</table>
+
-
Do it all again except B0031 (2005) because part length ~14 bp.  C0056 ~ 800 bp when it should be 636 bp and I13507 had really fat bands.
+
'''To-Do List:'''
-
 
+
-
To-Do List:
+
<ul>
<ul>
-
     <li>Purify I0500 (2005) at a higher concentrating (X3) (tape wells together)
+
     <li>Make o/n of Q03400 (2005) (2006)
-
     <li>Purify E0240 (2005) while testing part lengths of B0015 (2005), C0056 (2005) and I13507
+
     <li>Miniprep Q04400 (2005) (2006) and Q04510 (2005) (2006)
-
        (2005) again
+
     <li>Replate UT1 with miniprep from a good conlony
-
     <li>Check compatibilities with cI plasmids and I12006 + E0240
+
     <li>Miniprep Q04400 (2005) (2006) Q04510 (2005) (2006) and check to see if bands are correct.  If
-
     <li>Ligate Parts I0500 (2005), J06801 (2006), E0240 (2005)
+
        so, digest with EcoRI/XbaI and gel extract
 +
    <li>Ligate with UT1
</ul>
</ul>
-
== September 6, 2006 ==
+
[http://2006.igem.org/University_of_Toronto_2006 Home]
-
We started the mini-prep procedure for B0015 (2005) and I13507 (2005) by preparing an o/n growth of cells.  We then prepared freezer stock of J06801 (2006), B0034 (2005), C0056 (2005), E0240 (2005), I0500 (2005), I12006 (2005).
+
== October 17, 2006 ==
-
We began the ligation procedure using I0500 (2005) and J06801 (2006).  We were only able to get J06801 to work so we proceeded with the quantitation of that part only.  We obtained 3.0 ng/uL of purified J06801 DNA ready to be ligated tomorrow.
+
'''Melinda:'''
 +
<ul>
 +
    <li>Double Digetsed J04450 (W), UT1 DEFG with XbaI/SpeI using Buffer 2
 +
    <li>Results:
 +
    <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>
-
To-Do List:
+
'''Note – Change in protocol:'''
<ul>
<ul>
-
     <li>Repeat ligation attempt using I0500 (2005) and maybe continue with E0240 (2005)
+
     <li>After incubation for 1 hour, centrifuge tubes for 5 min @ 3000 RPM to pellet cells
-
    <li>If plasmids are compatible, we can ligate I12006 (2005) and E0240 (2004) and co-transform
+
     <li>Remove 800 uL of supernatant
-
        with a plasmid that expresses cI
+
     <li>Resuspend pellet with remaining 200 uL
-
     <li>Transform B0031 (2005) to make a freezer stock after 2 days.
+
     <li>Spread on plate and wait ~15-20 min.
-
     <li>Complete MP procedure for B0015 (2005), I13507 (2005)
+
-
     <li>Then check part/plasmid length, (with double digest), of B0031 (2005), C0056 (2005), B0015
+
-
        (2005), I13507 (2005)
+
</ul>
</ul>
-
== September 5, 2006 ==
+
[http://2006.igem.org/University_of_Toronto_2006 Home]
-
We tested the newly prepared MPs as well as two of the parts from yesterday using a double digest (XbaI and SpeI with Buffer 2).
+
== October 16, 2006 ==
-
<table border=1 cellspacing=1>
+
'''Melinda:'''
-
     <tr><th>Part Name<th>Part Description<th>Parts Match<th>Plasmid Match
+
<ul>
-
     <tr><td>J06801 a (2006)</td><td>Inverter with strong RBS</td><td>O</td><td>O
+
     <li>Checked parts, including the relevant parts from Waterloo (W):
-
    <tr><td>J06801 b (2006)</td><td>Inverter with strong RBS</td><td>O</td><td>O
+
     <ul>
-
    <tr><td>J06501 (2006)</td><td>Repressor, LacI temp sensitive</td><td>X</td><td>X
+
        <li>I0500 (W): (5000 – 4000, 1500 – 1000) – correct!
-
    <tr><td>B0034 (2005)</td><td>RBS (Elowitz 1999)</td><td>--</td><td>O
+
        <li>J04450 (W): (2500 – 2000) – plasmid is correct, but no part?
-
    <tr><td>C0056 (2005)</td><td>Repressor protein 434 cI</td><td>--</td><td>O
+
        <li>J06801 (W): (8000 – 6000) – not correct!
-
    <tr><td>E0240 (2005)</td><td>Medium RBS.GFP.Terminator</td><td>O</td><td>O
+
        <li>E0240 (W): (4000 – 3500, 3500 – 3000, 1000 – 750) – part is correct?
-
    <tr><td>E0422 (2005)</td><td>RBS.ECFP.Terminator</td><td>X</td><td>X
+
        <li>I12006 (W): (5000 – 4000, 2500 – 2000) - plasmid is correct, undigested plasmid?
-
</table>
+
        <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>
-
We transformed B0015 (2005) and I13507 (2005).
+
'''To-Do List:'''
 +
<ul>
 +
    <li>Repeat Double Digest of J04450 (W) and UT1 ABCD with XbaI/SpeI (Run undigested plasmids beside each double digest)
 +
    <li>Make more Amp plates
 +
    <li>If digest is successful, transform DH5a cells with the successful parts
 +
    <li>Find tetR replacements for cI and ligate those parts together.
 +
</ul>
-
To prepare a freezer stock, we made o/n of J06801 (2006), B0034 (2005), C0056 (2005), E0240 (2005), I0500 (2005), I12006 (2005)
+
[http://2006.igem.org/University_of_Toronto_2006 Home]
-
It now looks like we can attempt a ligation according to the first stage in the proposed CDR modules (I0500 + J06801 + E0240).
+
== October 15, 2006 ==
-
To-Do List for Tomorrow:
+
'''Charles:'''
<ul>
<ul>
-
     <li>MP B0015 (2005), I13507 (2005)
+
     <li>Made o/n of the newly plated UT2 2/4 and UT3 7 as well as UT2/UT3 freezer stock
-
    <li>Make a freezer stock of 06801 (2006), B0034 (2005), C0056 (2005), E0240 (2005), I0500 (2005), I12006 (2005)
+
-
    <li>Ligate I0500 (2005), J06801 (2006), and E0240 (2005)
+
</ul>
</ul>
-
== September 4, 2006 ==
+
'''To-Do List:'''
-
 
+
-
Started the mini-prep (MP) procedure (o/n growth) on E0240 (2005), B0034 (2005), J06801 (2006) and J06501 (2005)
+
-
 
+
-
Obtained a MP for C0056 (2005) and E0422 (2005).
+
-
 
+
-
Plan to do:
+
<ul>
<ul>
-
    <li>Check lengths of E0422 (2005) and C0056 (2005)
+
     <li>Make sure the cells fluoresce by diluting in IPTG
-
    <li>Transform B0015 (2005) as a backup/practice
+
-
    <li>MP B0034 (2005), E0240 (2005), J06501 (2006), and J06801 (2006) -- check lengths once done
+
-
     <li>Make I13507 (2005/2006) -- RFP (minus a promoter)
+
</ul>
</ul>
-
== September 3, 2006 ==
+
[http://2006.igem.org/University_of_Toronto_2006 Home]
-
 
+
-
We decided to recheck the more relevant parts using a double digest (XbaI and SpeI with Buffer 2).  This time we compared both the plasmid length and the actual part length.  Again O = match, X = no match and -- = inconclusive)
+
-
 
+
-
<table border=1 cellspacing=1>
+
-
    <tr><th>Part Name<th>Part Description<th>Parts Match<th>Plasmid Match
+
-
    <tr><td>I0500a (2005)</td><td>Promoter inducible by pBad/araC – part of thermometer</td><td>O
+
-
        </td><td>O
+
-
    <tr><td>I0500b (2005)</td><td>Promoter inducible by pBad/araC – part of thermometer</td><td>O
+
-
        </td><td>O
+
-
    <tr><td>J06801 (2006)</td><td>Inverter with strong RBS</td><td>O</td><td>O
+
-
    <tr><td>B0031 (2005)</td><td>Medium RBS</td><td>--</td><td>O
+
-
    <tr><td>B0031 (2006)</td><td>Medium RBS</td><td>X</td><td>X
+
-
    <tr><td>C0056 (2006)</td><td>DNA sequence for repressor protein 434 cI</td><td>X</td><td>X
+
-
    <tr><td>B0015 (2006)</td><td>Terminator</td><td>--</td><td>O
+
-
    <tr><td>J04450 (2006)</td><td>RFP switched off by IPTG</td><td>X</td><td>X
+
-
    <tr><td>I12006 (2005)</td><td>Modified lamdba Prm promoter (repressed by 434 cI)</td><td>--
+
-
        </td><td>O
+
-
    <tr><td>E0240 (2006)</td><td>Medium RBS.GFP.Terminator</td><td>X</td><td>X
+
-
</table>
+
-
 
+
-
Began min-prep procedure (o/n growth) for C0056 (2005) and E0422 (2005).
+
-
Transformed new parts from the 2005 DNA plate, (E0240, B0034, and J06501), and transformed more of the J06501 (2006) from the stock solution to produce more DNA for the mini-prep.
+
== October 14, 2006 ==
-
== September 2, 2006 ==
+
'''Charles, Jovan:'''
 +
<ul>
 +
    <li>Mini-prepped UT2 2/4 and UT3 7
 +
    <li>Transform UT2 2/4 and UT3 7 into DH5a-z1 and DH5a cells
 +
    <li>Continue with the IPTG test for the rest of the colonies that we weren’t able to test
 +
        yesterday
 +
</ul>
-
Now that we knew the enzymes are working, we proceeded to test the MiniPreps from 2005 and 2006 (O = match, X = no match):
+
'''To-Do List:'''
-
 
+
-
<table border=1 cellspacing=1>
+
-
    <tr><th> <th>Part Name<th>Part Description<th>Parts+Length<br>Match
+
-
    <tr><td rowspan=5>Gel 1</td><td>C0051 (2005)</td><td>Repressor, Lambda cI</td><td>X</td>
+
-
    <tr><td>E00433 (2005)</td><td>Reporter containing LacZ alpha fragment</td><td>O</td>
+
-
    <tr><td>B0031 (2005)</td><td>Medium RBS</td><td>O</td>
+
-
    <tr><td>R0040 (2005)</td><td>Promoter driven by tetR but action inhibited by aTc</td><td>O</td>
+
-
    <tr><td>R0011 (2005)</td>
+
-
        <td>Promoter regulated by lacI – part of BBa_J06801 (Inverter)</td><td>O</td>
+
-
    <tr><td rowspan=9>Gel 2</td><td>I0500 a (2005)</td>
+
-
        <td>Promoter inducible by pBad/araC – part of thermometer</td><td>O
+
-
    <tr><td>I0500 b (2005)</td><td>Promoter inducible by pBad/araC – part of thermometer</td><td>O
+
-
    <tr><td>B0031 (2006)</td><td>Medium RBS</td><td>X
+
-
    <tr><td>C0056 (2006)</td><td>DNA sequence for repressor protein 434 cI</td><td>X
+
-
    <tr><td>E0422 (2006)</td><td>RBS.ECFP.Terminator</td><td>X
+
-
    <tr><td>J04450 (2006)</td><td>RFP switched off by IPTG</td><td>X
+
-
    <tr><td>E0240 (2006)</td><td>Medium RBS.GFP.Terminator</td><td>X
+
-
    <tr><td>R0011 (2006)</td><td>Promoter regulated by lacI – part of BBa_J06801 (Inverter)</td><td>X
+
-
    <tr><td>E0433 (2006)</td><td>Reporter containing LacZ alpha fragment</td><td>O
+
-
</table>
+
-
 
+
-
We also began the transformation of some of the 2005 parts:
+
<ul>
<ul>
-
     <li>I0500 (Promoter inducible by pBad/araC – part of thermometer)
+
     <li>Measure absorbance spectrum of Arabinose
-
     <li>C0056 (DNA sequence for repressor protein 434 cI)
+
     <li>Make o/n of working UT2/UT3 for repeat of IPTG test.
-
     <li>I12006 (Modified lamda Prm promoter repressed by 434 cI)
+
     <li>Look into using tetR and tet pL instead of cI and Prm+
-
    <li>E0422 (RBS.ECFP.Terminator)
+
</ul>
</ul>
-
 
-
== September 1, 2006 ==
 
-
 
-
With a lot (a LOT) of help from Seema, we now have 24 Eppendorf tubes of competent cells (DH5a-z1) in the -80C freezer.  We also have ~10 plates with Ampicillin (Amp), ~8 plates with Kanamycin (Kan), and 4 plates with Amp/Kan resistance.  Seema also sorted out our iGEM box and separated between the 2005 parts and the 2006 parts as well as grouped the enzymes, reagents, and ladders.
 
-
 
-
Due to a lot of problems with previous attempts at transformations and quantifications, we decided to begin testing the enzymes as well all the part lengths so that we can get a handle of what we have to work with.  So we tested all the enzymes, (EcoRI, XbaI, SpeI, and PstI) using a test plasmid.  In theory, the uncut plasmid should move farther in the gel than the cut plasmid because the circular plasmid is more compact and would better be able to move through the agarose gel.  This was indeed the case and all the enzymes cut successfully as shown the by the 4 bands being at the same height above the uncut band.
 
-
 
-
One thing to note was that the enzyme Xba1 worked slower than the others.  Since the idea was to just check if the enzymes were working, we didn’t need to let the enzymes cut for the full 1 hr (as per the protocol).  Thus, we ran the gel about 40 min early and saw two bands (one for each of the cut and uncut plasmid) in the Xba1 lane.
 
-
 
-
== August, 11, 2006 ==
 
-
 
-
Hey team,
 
-
 
-
It looks like we'll soon be underway and get to start building and testing parts.  I seem to remember Farshid saying he and Jessica already extracted the parts that we needed from the registry, so that we can move on from there.  There's a semi-final outline of what we'd like to do on the Critical Design Review (CDR) page so take a look at that.  Once we get approval from Prof. Davies, we can get started.
 
-
 
-
The overall idea is to "assign" a part to a member so that member is responsible for growing/maintaining that part as well as adding that part to someone else's part.  Also, depending on other people's thoughts on this, it would be a good idea to double up on the parts so that we get at least two cracks at making a part at the same time.  This allows everyone to participate in the process from start to finish.  Of course there will be a lot of flexibility in who does what, but this gives us some sort of baseline to start with.  I'll be making some sort of sign-up sheet so if you want to make a specific part for some reason you can.  I was thinking of using Google calendar to help with the coordination, so if you want a Gmail address, you can just email me and I can invite you (Gmail is actually really good).  I'll keep you updated on this idea though.
 
-
 
-
Finally, <b>CHECK THE WIKI REGULARLY!!</b>  We are now going to start doing stuff so there should be regular postings on the Wiki and so I don't want to flood everyone's inboxes.  I will try to see if there is some way to indicate whether a link has been modified in some way so that you know there has been an update.  Or if any of you know how to implement that by all means go for it.
 
-
 
-
Charles Yoon
 
[http://2006.igem.org/University_of_Toronto_2006 Home]
[http://2006.igem.org/University_of_Toronto_2006 Home]

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

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

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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