Construction

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== September 11, 2006 ==
+
<center>
 +
<font size=4>< [[Aug11-Sep7]] | [[Sep8-14]] | [[Sep15-26]] | [[Sep27-Oct3]] | [[Oct4-Oct13]] | [[Construction]] ></font>
 +
</center>
 +
== October 30, 2006 ==
 +
'''Charles:'''
 +
<ul>
 +
    <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>
-
== September 10, 2006 ==
+
'''To-Do List:'''
 +
<ul>
 +
    <li>Make more M9 media
 +
    <li>Make competent cells
 +
    <li>Ligate final parts together
 +
</ul>
-
Miniprepped a lot of parts:
+
== October 29, 2006 ==
-
<table border=1 cellspacing=1>
+
-
    <tr><th>Part Name<th>Parts Match<th>Plasmid Match
+
-
    <tr><td>I0500 C (2005)</td><td>X</td><td>O
+
-
    <tr><td>I0500 D (2005)</td><td>X</td><td>O
+
-
    <tr><td>E0240 C (2005)</td><td>O</td><td>O
+
-
    <tr><td>E0240 D (2005)</td><td>O</td><td>O
+
-
    <tr><td>B0031 C (2005)</td><td>--</td><td>O
+
-
    <tr><td>B0031 B (2005)</td><td>--</td><td>O
+
-
    <tr><td>I12006 D (2005)</td><td>X</td><td>X
+
-
    <tr><td>I12006 C (2005)</td><td>X</td><td>X
+
-
    <tr><td>I13507 B (2005)</td><td>O</td><td>O
+
-
    <tr><td>I13507 C (2005)</td><td>O</td><td>O
+
-
    <tr><td>J06801 D (2006)</td><td>O</td><td>O
+
-
    <tr><td>J06801 C (2006)</td><td>O</td><td>O
+
-
    <tr><td>C0056 B (2005)</td><td>X</td><td>O
+
-
    <tr><td>C0056 C (2005)</td><td>X</td><td>O
+
-
    <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
+
'''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>
-
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)
+
== October 28, 2006 ==
-
To-Do List:
+
'''Charles:'''
<ul>
<ul>
-
     <li>miniprep B0015 (2005/6) and I0500 (2005)
+
     <li>Mini-prepped UT9, UT10, UT11 ABC (may be slightly messed up – miniprep again?)
-
     <li>prepare o/n growth of new part
+
    <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>
-
== September 9, 2006 ==
+
'''To-Do List:'''
 +
- Make o/n of DH5a and DH5a-z1
 +
- Miniprep pBAD AB
 +
- Make Amp and Kan plates
-
Mini-prepped everything (from yesterday)
+
== October 27, 2006 ==
-
Plated B0015 (2005) and B0015 (2006)
+
-
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)
+
-
== September 8, 2006 ==
+
'''Charles, Nick:'''
 +
<ul>
 +
    <li>M9 Test – 0% - 2% Arabinose in UT3 and DH5a (control), temperature test at 0.2% Arabinose,
 +
        IPTG verification at 0% and 2% arabinose:
 +
    <ul>
 +
        <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>
-
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.
+
'''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>
-
<table border=1 cellspacing=1>
+
== October 26, 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
+
'''Andy, Natalie, Charles, Tara, HoKwon:'''
 +
<ul>
 +
    <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>
-
The quantitation results are as follows:
+
'''To-do list:'''
<ul>
<ul>
-
     <li>E0240 B, 8.0 ng/uL
+
     <li>Make o/n of UT9, UT10, UT11 for miniprep
-
     <li>E0240 A, 6.0 ng/uL
+
     <li>Ligate I0500 (2005) with I13504 (2005/2006)
-
     <li>I0500 B, 13.5 ng/uL
+
     <li>M9 testing of UT3
</ul>
</ul>
-
To-Do List:
+
== October 25, 2006 ==
 +
 
 +
'''Andy:'''
<ul>
<ul>
-
     <li>transform cells with B0015 (2005)
+
     <li>Minipreppred UT6, UT7, UT8
-
     <li>MP B0031 (2005), B0034 (2005), C0056 (2005), E0240 (2005), I0500 (2005), I12006 (2005),  
+
     <li>Gel-extracted (only for correct bands)
-
        I13507 (2005), J06801 (2006)
+
          <ul>         
-
     <li>Ligate I0500 + J06801
+
              <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>
-
== September 7, 2006 ==
+
'''To-do list:'''
 +
<ul>
 +
    <li>Quantitate and ligate UT6, UT7 with I13504 (2005/2006)
 +
    <li>Quantitate and ligate R0011 (2005) with I13507 (2005)
 +
    <li>M9 testing of UT2, UT3
 +
</ul>
-
Obtained purified DNA of I0500 (2005) with a concentration of 0 ng/uL.  Try again tomorrow
+
== October 24, 2006 ==
-
Made mini-preps of B0015 (2005) and I13507 (2005) and made a plate of B0031 (2005)
+
'''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>
-
<table border=1 cellspacing=1>
+
== October 23, 2006 ==
-
    <tr><th>Part Name<th>Part Description<th>Parts Match<th>Plasmid Match
+
-
    <tr><td>B0015 (2005)</td><td>Terminator</td><td>--</td><td>--
+
-
    <tr><td>B0031 (2005)</td><td>Medium RBS</td><td>--</td><td>O
+
-
    <tr><td>C0056 (2005)</td><td>Repressor protein 434 cI</td><td>--</td><td>O
+
-
    <tr><td>I13507 (2005)</td><td>RBS.mRFP.Terminator</td><td>--</td><td>--
+
-
</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.
+
'''Andy:'''
 +
<ul>
 +
    <li>Gel-extrated I13507 (2005) X/P
 +
    <li>Made o/n of I13522 (2006), I13504 (2005/2006) x 2, R0011 (2005) for minipreps
 +
    <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>
-
To-Do List:
+
'''To-do List:'''
<ul>
<ul>
-
     <li>Purify I0500 (2005) at a higher concentrating (X3) (tape wells together)
+
     <li>Quantitate and Ligate R0011 (2005) with I13507 (2005)
-
     <li>Purify E0240 (2005) while testing part lengths of B0015 (2005), C0056 (2005) and I13507
+
     <li>Miniprep and length-check I13522 (2006), I13504 (2005/2006) x 2, R0011 (2005)
-
        (2005) again
+
     <li>Make multiple o/n of UT6-8 for miniprep
-
     <li>Check compatibilities with cI plasmids and I12006 + E0240
+
-
    <li>Ligate Parts I0500 (2005), J06801 (2006), E0240 (2005)
+
</ul>
</ul>
-
== September 6, 2006 ==
+
== October 22, 2006 ==
-
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).
+
'''Massive To-do List'''
-
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.
+
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>Repeat ligation attempt using I0500 (2005) and maybe continue with E0240 (2005)
+
     <li>Transform and miniprep I13522 (2006) (amp) from registry DNA-2 15H
-
     <li>If plasmids are compatible, we can ligate I12006 (2005) and E0240 (2004) and co-transform
+
     <li>Transform and miniprep I13504 (2005/2006) (amp) from registry DNA-1 12D (2005/2006)
-
        with a plasmid that expresses cI
+
    <li><font color=red>Miniprep</font> 2 UT1 for ligation (not urgent)
-
     <li>Transform B0031 (2005) to make a freezer stock after 2 days.
+
     <li>Ligate UT1 with Q03400, Q04400 (2005/2006) (4 ligations) (Name UT6-9)
-
     <li>Complete MP procedure for B0015 (2005), I13507 (2005)
+
     <li>If possible, <font color=red>ligate</font> R0011 (2005) with I13507 (2005) in parallel (Name UT10)
-
     <li>Then check part/plasmid length, (with double digest), of B0031 (2005), C0056 (2005), B0015
+
     <li><font color=red>Ligate</font> UT6-9 with I13504 (4 ligations) (Name UT11-14)
-
        (2005), I13507 (2005)
+
    <li><font color=red>Ligate</font> UT11-14 with UT10 (4 ligations) (Nam UT15-18)
</ul>
</ul>
-
== September 5, 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>
-
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).
+
'''Andy:'''
 +
<ul>
 +
    <li>Gel-extrated R0011 (2005) S/P (band: 2000 - correct)
 +
    <li>Cannot gel-extract I13507 (2005) X/P because there was no more extraction column (bands: 2500-3000, 2000-2500, 750-1000 - correct)
 +
    <li>Transformed R0011 (2005), I13522 (2006), I13504 (2005/2006)
 +
    <li>Quantitated
 +
        <ul>
 +
        <li>Q3400 (2005) E/X (no band)
 +
        <li>Q4400 (2005) E/X (band: 5000 - correct 120 ng/uL)
 +
        <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>
 +
    <li>o/n of UT3 x 5, UT2 x 1, and DH5a x 4 for temperature, arabinose, and plate testing
 +
</ul>
-
<table border=1 cellspacing=1>
+
'''To-do List:'''
-
    <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>Ligate UT1 with Q3400 (2006), Q4400 (2005/2006)
-
    <tr><td>J06801 b (2006)</td><td>Inverter with strong RBS</td><td>O</td><td>O
+
     <li>Make o/n of I13522 (2006) for miniprep and plate-test, I13504 (2005/2006), I13507 (2005) for miniprep
-
     <tr><td>J06501 (2006)</td><td>Repressor, LacI temp sensitive</td><td>X</td><td>X
+
     <li>Gel-extract I13507 (2005) X/P
-
    <tr><td>B0034 (2005)</td><td>RBS (Elowitz 1999)</td><td>--</td><td>O
+
     <li>Temperature, arabinose, and plate testing
-
     <tr><td>C0056 (2005)</td><td>Repressor protein 434 cI</td><td>--</td><td>O
+
     <li>Purchase gel-extraction kit, make Amp plates
-
     <tr><td>E0240 (2005)</td><td>Medium RBS.GFP.Terminator</td><td>O</td><td>O
+
</ul>
-
     <tr><td>E0422 (2005)</td><td>RBS.ECFP.Terminator</td><td>X</td><td>X
+
-
</table>
+
-
We transformed B0015 (2005) and I13507 (2005).
+
'''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>
-
To prepare a freezer stock, we made o/n of J06801 (2006), B0034 (2005), C0056 (2005), E0240 (2005), I0500 (2005), I12006 (2005)
+
'''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>
-
It now looks like we can attempt a ligation according to the first stage in the proposed CDR modules (I0500 + J06801 + E0240).
 
-
To-Do List for Tomorrow:
+
[http://2006.igem.org/University_of_Toronto_2006 Home]
 +
 
 +
== October 19, 2006 ==
 +
 
 +
'''Melinda:'''
<ul>
<ul>
-
     <li>MP B0015 (2005), I13507 (2005)
+
     <li>Miniprepped Q04400 (2005/2006) and Q04510 (2005/2006)
-
     <li>Make a freezer stock of 06801 (2006), B0034 (2005), C0056 (2005), E0240 (2005), I0500 (2005), I12006 (2005)
+
     <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>Ligate I0500 (2005), J06801 (2006), and E0240 (2005)
+
</ul>
</ul>
-
== September 4, 2006 ==
+
'''To-Do List:'''
 +
<ul>
 +
    <li>Miniprep Q03400 (2005/2006)
 +
    <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>
-
Started the mini-prep (MP) procedure (o/n growth) on E0240 (2005), B0034 (2005), J06801 (2006) and J06501 (2005)
+
[http://2006.igem.org/University_of_Toronto_2006 Home]
-
Obtained a MP for C0056 (2005) and E0422 (2005).
+
== October 18, 2006 ==
-
Plan to do:
+
'''Andy, Konstantin:'''
<ul>
<ul>
-
     <li>Check lengths of E0422 (2005) and C0056 (2005)
+
     <li>Transformed and plated Q03400 (2005) (2006)
-
     <li>Transform B0015 (2005) as a backup/practice
+
     <li>Gel-extracted UT1 DEF with EcoRI/SpeI (2500-3000, 5000, 8000), 8000 band bright, digestion appeared incomplete
-
     <li>MP B0034 (2005), E0240 (2005), J06501 (2006), and J06801 (2006) -- check lengths once done
+
     <li>Made A, K, AK plates
-
     <li>Make I13507 (2005/2006) -- RFP (minus a promoter)
+
    <li>Made o/n of Q04400 (2005) (2006) Q04510 (2005) (2006)
 +
     <li>Tested UT3 in DH5a with 0%-2% arabinose
</ul>
</ul>
-
== September 3, 2006 ==
+
'''To-Do List:'''
 +
<ul>
 +
    <li>Make o/n of Q03400 (2005) (2006)
 +
    <li>Miniprep Q04400 (2005) (2006) and Q04510 (2005) (2006)
 +
    <li>Replate UT1 with miniprep from a good conlony
 +
    <li>Miniprep Q04400 (2005) (2006) Q04510 (2005) (2006) and check to see if bands are correct.  If
 +
        so, digest with EcoRI/XbaI and gel extract
 +
    <li>Ligate with UT1
 +
</ul>
-
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)
+
[http://2006.igem.org/University_of_Toronto_2006 Home]
-
<table border=1 cellspacing=1>
+
== October 17, 2006 ==
-
    <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).
+
'''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>
-
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.
+
'''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>
-
== September 2, 2006 ==
+
[http://2006.igem.org/University_of_Toronto_2006 Home]
-
Now that we knew the enzymes are working, we proceeded to test the MiniPreps from 2005 and 2006 (O = match, X = no match):
+
== October 16, 2006 ==
-
<table border=1 cellspacing=1>
+
'''Melinda:'''
-
    <tr><th> <th>Part Name<th>Part Description<th>Parts+Length<br>Match
+
<ul>
-
     <tr><td rowspan=5>Gel 1</td><td>C0051 (2005)</td><td>Repressor, Lambda cI</td><td>X</td>
+
     <li>Checked parts, including the relevant parts from Waterloo (W):
-
     <tr><td>E00433 (2005)</td><td>Reporter containing LacZ alpha fragment</td><td>O</td>
+
     <ul>
-
    <tr><td>B0031 (2005)</td><td>Medium RBS</td><td>O</td>
+
        <li>I0500 (W): (5000 – 4000, 1500 – 1000) – correct!
-
    <tr><td>R0040 (2005)</td><td>Promoter driven by tetR but action inhibited by aTc</td><td>O</td>
+
        <li>J04450 (W): (2500 – 2000) – plasmid is correct, but no part?
-
    <tr><td>R0011 (2005)</td>
+
         <li>J06801 (W): (8000 – 6000) – not correct!
-
         <td>Promoter regulated by lacI – part of BBa_J06801 (Inverter)</td><td>O</td>
+
         <li>E0240 (W): (4000 – 3500, 3500 – 3000, 1000 – 750) – part is correct?
-
    <tr><td rowspan=9>Gel 2</td><td>I0500 a (2005)</td>
+
        <li>I12006 (W): (5000 – 4000, 2500 – 2000)  - plasmid is correct, undigested plasmid?
-
         <td>Promoter inducible by pBad/araC – part of thermometer</td><td>O
+
        <li>UT2 2: (3500 – 3000, 2500 – 2000, 2000 – 1500) – correct, extra band?
-
    <tr><td>I0500 b (2005)</td><td>Promoter inducible by pBad/araC – part of thermometer</td><td>O
+
        <li>UT2 4: (3500 – 3000, 2500 – 2000, 2000 – 1500) – correct, extra band?
-
    <tr><td>B0031 (2006)</td><td>Medium RBS</td><td>X
+
        <li>UT3 7: (3500 – 3000, 2500 – 2000) – correct!
-
    <tr><td>C0056 (2006)</td><td>DNA sequence for repressor protein 434 cI</td><td>X
+
     </ul>
-
    <tr><td>E0422 (2006)</td><td>RBS.ECFP.Terminator</td><td>X
+
     <li>Viewed cells induced by IPTG under microscope and verified the functionality of UT2/UT3 in DH5a and DH5a-z1
-
    <tr><td>J04450 (2006)</td><td>RFP switched off by IPTG</td><td>X
+
</ul>
-
     <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:
+
'''To-Do List:'''
<ul>
<ul>
-
     <li>I0500 (Promoter inducible by pBad/araC – part of thermometer)
+
     <li>Repeat Double Digest of J04450 (W) and UT1 ABCD with XbaI/SpeI (Run undigested plasmids beside each double digest)
-
     <li>C0056 (DNA sequence for repressor protein 434 cI)
+
     <li>Make more Amp plates
-
     <li>I12006 (Modified lamda Prm promoter repressed by 434 cI)
+
     <li>If digest is successful, transform DH5a cells with the successful parts
-
     <li>E0422 (RBS.ECFP.Terminator)
+
     <li>Find tetR replacements for cI and ligate those parts together.
</ul>
</ul>
-
== September 1, 2006 ==
+
[http://2006.igem.org/University_of_Toronto_2006 Home]
-
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.
+
== October 15, 2006 ==
-
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.
+
'''Charles:'''
 +
<ul>
 +
    <li>Made o/n of the newly plated UT2 2/4 and UT3 7 as well as UT2/UT3 freezer stock
 +
</ul>
-
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.
+
'''To-Do List:'''
 +
<ul>
 +
    <li>Make sure the cells fluoresce by diluting in IPTG
 +
</ul>
-
== August, 11, 2006 ==
+
[http://2006.igem.org/University_of_Toronto_2006 Home]
-
Hey team,
+
== October 14, 2006 ==
-
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.
+
'''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>
-
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.
+
'''To-Do List:'''
-
 
+
<ul>
-
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.
+
    <li>Measure absorbance spectrum of Arabinose
-
 
+
    <li>Make o/n of working UT2/UT3 for repeat of IPTG test.
-
Charles Yoon
+
    <li>Look into using tetR and tet pL instead of cI and Prm+
 +
</ul>
[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.


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

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

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

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

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

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.

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

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.

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

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+

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

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