Team:TMU-Tokyo/Notebook experiment/4th

From 2013.igem.org

(Difference between revisions)
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Assay<Br>
Assay<Br>
       Pythagorean devices
       Pythagorean devices
-
<Table>
+
<style>
-
<Tr>
+
table {
-
<Td>LB 2ml + arabinose -10%  0.1ml</Td>
+
border-collapse: collapse;
-
<Td> + with the inoculating loop 1655 ins①~③ No.1<Br>
+
}
-
      + with the inoculating loop 1655 ins①~③ No.2<Br>
+
th {
-
      + with the inoculating loop 1655 ins①~③ No.3<Br>
+
border: solid 1px #666666;
-
      + with  1655</Td>
+
color: #000000;
-
</Tr>
+
background-color: #ff9999;
-
<Tr>
+
}
-
<Td>LB 2ml                      </Td>
+
td {
-
<Td>+ with the inoculating loop 1655 ins①~③ No.1<Br>
+
border: solid 1px #666666;
-
    + with the inoculating loop 1655 ins①~③ No.2<Br>
+
color: #000000;
-
    + with the inoculating loop 1655 ins①~③   No.3<Br>
+
background-color: #ffffff;
-
                              +   with            1655</Td>
+
}
-
</Tr>
+
</style>
-
</Table>
+
<table>
-
                    ⇒I culture it at 39 degrees for 2h.
+
<thead>
-
 
+
<tr>
-
 
+
<th colspan="5">
-
 
+
<PCR CHECK preculture>
-
 
+
</th>
-
 
+
</tr>
-
 
+
</thead>
-
 
+
<tbody>
-
 
+
<tr>
-
 
+
<th rowspan="2" colspan="9">
 +
      Pythagorean devices
 +
</th>
 +
</tr>
 +
<tr>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
LB 2ml + arabinose -10%  0.1ml + with the inoculating loop 1655 ins①~③ No.1
 +
</th>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
                              +                                    No.2
 +
</th>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
                              +                                    No.3
 +
</th>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
1655
 +
</th>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
LB 2ml                      + with the inoculating loop 1655 ins①~③ No.1
 +
</th>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
                              +                                      No.2
 +
</th>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
                              +                                     No.3
 +
</th>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
1655
 +
</th>
 +
</tr>
 +
<tr>
 +
<th rowspan="2" colspan="9">
 +
                    ⇒I culture it at 39 degrees for 2h.
 +
</th>
 +
</tr>
 +
<tr>
 +
</tr>
 +
<tr>
 +
<th rowspan="5" colspan="5">
 +
<PCR CHECK Pythagorean devices FRT>
 +
</th>
 +
</tr>
 +
<tr>
 +
<td>
 +
Cell suspension
 +
</td>
 +
<td>
 +
ins①-③
 +
</td>
 +
<td colspan="3">
 +
ara(+) No.1~3
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
Ins①-③
 +
</td>
 +
<td colspan="3">
 +
ara(-) No.1~3
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
1655
 +
</td>
 +
<td colspan="3">
 +
ara(+)
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
1655
 +
</td>
 +
<td colspan="2">
 +
ara(-)
 +
</td>
 +
</tr>
 +
<tr>
 +
<th rowspan="4">
 +
Premix(1)
 +
</th>
 +
<td>
 +
GXL Buff
 +
</td>
 +
<td colspan="3">
 +
20
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
dNTP
 +
</td>
 +
<td colspan="3">
 +
8
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
GXL
 +
</td>
 +
<td colspan="4">
 +
2
 +
</td>
 +
</tr>
 +
<tr>
 +
<td colspan="3">
 +
-30
 +
</td>
 +
</tr>
 +
<tr>
 +
<th rowspan="4">
 +
Premix(2)
 +
</th>
 +
<td>
 +
Premix(1)
 +
</td>
 +
<td colspan="3">
 +
27
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
CmNR-not2
 +
</td>
 +
<td colspan="3">
 +
9
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
141-1
 +
</td>
 +
<td colspan="4">
 +
9
 +
</td>
 +
</tr>
 +
<tr>
 +
<td colspan="3">
 +
-45
 +
</td>
 +
</tr>
 +
<tr>
 +
<th rowspan="4">
 +
mix
 +
</th>
 +
<td>
 +
Cell suspension
 +
</td>
 +
<td colspan="3">
 +
5λ
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
Premix(2)
 +
</td>
 +
<td colspan="4">
 +
5λ
 +
</td>
 +
</tr>
 +
<tr>
 +
<td colspan="7">
 +
<4P3-3>
 +
</td>
 +
</tr>
 +
<tr>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
<PCR CHECK Ptet inversion>
 +
</th>
 +
</tr>
 +
<tr>
 +
<th rowspan="2">
 +
Cell suspension
 +
</th>
 +
<td>
 +
1655
 +
</td>
 +
<td>
 +
ins①~③
 +
</td>
 +
<td>
 +
ara(+)
 +
</td>
 +
<td>
 +
No.1~3
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
1655
 +
</td>
 +
<td>
 +
ins①~③
 +
</td>
 +
<td>
 +
ara(-)
 +
</td>
 +
<td>
 +
No.1~3
 +
</td>
 +
</tr>
 +
<tr>
 +
<th rowspan="4">
 +
Premix①
 +
</th>
 +
<td>
 +
GXL Buffer
 +
</td>
 +
<td colspan="3">
 +
30
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
dNTP
 +
</td>
 +
<td colspan="3">
 +
12
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
GXL
 +
</td>
 +
<td colspan="4">
 +
3
 +
</td>
 +
</tr>
 +
<tr>
 +
<td colspan="3">
 +
-45
 +
</td>
 +
</tr>
 +
<tr>
 +
<th rowspan="6" colspan="2">
 +
Premix②
 +
</th>
 +
<td>
 +
<Ptet1>
 +
</td>
 +
<td colspan="2">
 +
<Ptet2>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
Premix(1)
 +
</td>
 +
<td>
 +
21
 +
</td>
 +
<td colspan="2">
 +
21
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
FimE-C2-EcoRI
 +
</td>
 +
<td>
 +
7
 +
</td>
 +
<td colspan="2">
 +
7
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
TetP1
 +
</td>
 +
<td colspan="3">
 +
7
 +
</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">
 +
TetP2
 +
</td>
 +
<td colspan="3">
 +
7
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
-35
 +
</td>
 +
<td colspan="2">
 +
-35
 +
</td>
 +
</tr>
 +
<tr>
 +
<th rowspan="4">
 +
mix
 +
</th>
 +
<td>
 +
Premix②
 +
</td>
 +
<td colspan="3">
 +
5
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
Cell suspension
 +
</td>
 +
<td colspan="3">
 +
5
 +
</td>
 +
</tr>
 +
<tr>
 +
<td colspan="8">
 +
<4P3-3>
 +
</td>
 +
</tr>
 +
<tr>
 +
</tr>
 +
<tr>
 +
<th rowspan="2" colspan="9">
 +
<plating assay Pythagorean devices>
 +
</th>
 +
</tr>
 +
<tr>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
<plating assay Pythagorean devices>
 +
</th>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
LB + arabinose (0.2%) → plating (single colony isolation)
 +
</th>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
                          ・1655 + ins①~③     No.1~3
 +
</th>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
                          ・1655              No.1~3
 +
</th>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
LB + IPTG (0.4g/ml)  → plating (single colony isolation)
 +
</th>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
                          ・1655 + ins①~③    No.1~3
 +
</th>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
                          ・1655              No.1~3
 +
</th>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
LB                  → plating (single colony isolation)
 +
</th>
 +
</tr>
 +
<tr>
 +
<th colspan="5">
 +
                          ・1655 + ins①~③    No.1~3
 +
</th>
 +
</tr>
 +
<tr>
 +
<th rowspan="3" colspan="13">
 +
                          ・1655              No.1~3
 +
</th>
 +
</tr>
 +
<tr>
 +
</tr>
 +
<tr>
 +
</tr>
 +
</tbody>
 +
</table>

Revision as of 00:20, 28 September 2013



Experiment > 4th week

September 22th
Mini prep following plasmid.
fimE-pBAD(in pSB1A3), pSB1C3-Km, pSB1A3-Km
Result.

1: pSB1C3-Km, 2-4: pSB1A3-Km, 5-6: fimE-pBAD


Check PCR.
Fragment(control)Fragment 3 - ApCm
Primer141-49
SaApL		141-49
SaApL
TempleteEscherichia coli
MG1655 red
(Cell suspention)		(Cell suspention)
Result.

1-9: Fragment 3 - ApCm

Check PCR.
Fragment(control)Fragment 3 - ApCmFragment 3 - ApCm
Primer2 sets of the others141-1
141-20		141-1
141-74
TempleteEscherichia coli
MG1655 red
(Cell suspention)		(Cell suspention)
Result.

1-7: 141-1 and 141-20, 8: control(141-1 and 141-20),
9-15: 141-1 and 141-74, 16: control(141-1 and 141-74),


Digestion following plasmid.
pSB1C3-Km-Blunt


P1 Preparation following fragment.
Fragment 1, 2


September 23th
PCR following fragment.
FragmentFragment 3 - ApCm
PrimermhpA-CmC6
SaApL
TempleteFragment 3 - Cm
Ap(SaApR-SaApL)
Result.

1: Fragment 3 - Cm, 2: Fragment 3 - ApCm,
3: pSB1C3-Km-Blunt


Check PCR.
FragmentFragment 3 - Ap
(control)		Fragment 3 - ApCm
PrimerCmNR-Not
SaApL		CmNR-Not
SaApL
Templete(Cell suspention)(Cell suspention)
Result.

1-31: Fragment 3 - ApCm


Electro Pration
Fragment 3 - Cm to Escherichia coli MG1655 red.


P1 Preparation
Fragment 3 - ApCm


September 24th
P1 transdunction
Fragment 1, 2
Result.



FragmentFragment 3 - Ap
(control)		Fragment 3 - ApCm
PrimerCmNR-Not
141-1		CmNR-Not
141-1
Templete(Cell suspention)(Cell suspention)
Result.

1-16: Fragment 3 - ApCm


P1 transduction
Fragment 2 to Fragment 1
Fragment 1 to Fragment 2
Result.


Get Fragment 1-2

Electro Poration
Fragment 1, 2, 3 - Ap, 3 - Cm, 3 - ApCm (in pSBC3-Km) to Escherichia coli DH1
Result.



September 25th
Check PCR.
FragmentFragment 3 - ApCm
(control)		Fragment 3 - ApCm
Primer141-49
141-1		CmNR-Not
141-1
Templete(Cell suspention)(Cell suspention)
Result.

1-7: 141-49 and 141-1, 8: control(141-49 and 141-8),
9-15: 141-1 and CmNR-Not, 16: control(141-1 and CmNR-Not)


September 26th
P1 transduction
Fragment 3 to Fragment 1-2
Get Fragment 1-2-3.


Check PCR
FragmentFragment 1-2-3Fragment 1-2
(control)
PrimerCmNR-Not
141-1		141-49
141-1
Templete(Cell suspention)(Cell suspention)
Result.

1-7: Fragment 1-2-3, 8: cotrol, 9-10: Fragment 1,
11-13: Fragment 2, 14-15: Fragment 3 - Ap, 16-17: Fragment 3 - Cm
18-19: Fragment 3 - AmCm


September 27th
Assay
Pythagorean devices
Pythagorean devices
LB 2ml + arabinose -10% 0.1ml + with the inoculating loop 1655 ins①~③ No.1
+ No.2
+ No.3
1655
LB 2ml + with the inoculating loop 1655 ins①~③ No.1
+ No.2
+ No.3
1655
⇒I culture it at 39 degrees for 2h.
Cell suspension ins①-③ ara(+) No.1~3
Ins①-③ ara(-) No.1~3
1655 ara(+)
1655 ara(-)
Premix(1) GXL Buff 20
dNTP 8
GXL 2
-30
Premix(2) Premix(1) 27
CmNR-not2 9
141-1 9
-45
mix Cell suspension 5λ
Premix(2) 5λ
<4P3-3>
Cell suspension 1655 ins①~③ ara(+) No.1~3
1655 ins①~③ ara(-) No.1~3
Premix① GXL Buffer 30
dNTP 12
GXL 3
-45
Premix②
Premix(1) 21 21
FimE-C2-EcoRI 7 7
TetP1 7
TetP2 7
-35 -35
mix Premix② 5
Cell suspension 5
<4P3-3>
LB + arabinose (0.2%) → plating (single colony isolation)
・1655 + ins①~③ No.1~3
・1655 No.1~3
LB + IPTG (0.4g/ml) → plating (single colony isolation)
・1655 + ins①~③ No.1~3
・1655 No.1~3
LB → plating (single colony isolation)
・1655 + ins①~③ No.1~3
・1655 No.1~3


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