Team:TMU-Tokyo/Notebook experiment/4th

From 2013.igem.org

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<Hr>
<Hr>
-
<b>September 2nd</b><Br>
+
<b>September 22th</b><Br>
-
PCR following fragment. (1st PCR)<Br>
+
Mini prep following plasmid.<Br>
-
 
+
<Table><Tr><Td>fimE-pBAD(in pSB1A3), pSB1C3-Km, pSB1A3-Km</Td></Tr></Table>
 +
Result.<Br>
 +
<IMG Src="https://static.igem.org/mediawiki/2013/4/4c/TMU-Tokyo_0922.jpg"><Br>
 +
1: pSB1C3-Km, 2-4: pSB1A3-Km, 5-6: fimE-pBAD<Br>
 +
<Br>
 +
<Br>
 +
Check PCR.<Br>
<div style="text-align:center;">
<div style="text-align:center;">
<Table cellpadding="5">
<Table cellpadding="5">
<Tr>
<Tr>
-
<Td>Fragment</Td><Td>PtetR1</Td><Td>PtetR2</Td><Td>FLP</Td><Td>Sm</Td><Td>araC-P</Td>
+
<Td>Fragment</Td><Td>(control)</Td><Td>Fragment 3 - ApCm</Td>
</Tr>
</Tr>
<Tr>
<Tr>
-
<Td>Primer</Td><Td>CmNR-stu<Br>FLP-IRR/IRL</Td><Td>CmCR-stu<Br>FLP-IRR/IRL</Td><Td>FLP-L<Br>FLP-R</Td><Td>FLP-SaApL<Br>CmC</Td><Td>acaC-2<Br>BAD-C3</Td>
+
<Td>Primer</Td><Td>141-49<Br>SaApL</Td><Td>141-49<Br>SaApL</Td>
</Tr>
</Tr>
<Tr>
<Tr>
-
<Td>Templete</Td><Td>IRR/IRL-GFP<Br>(BBa_K880002)</Td><Td>IRR/IRL-GFP<Br>(BBa_K880002)</Td><Td>pFTGT</Td><Td>m(C2-Ap)Sm</Td><Td>pBAD/Myc-HisA</Td>
+
<Td>Templete</Td><Td><i>Escherichia coli</i><Br>MG1655 red<Br>(Cell suspention)</Td><Td>(Cell suspention)</Td>
</Tr>
</Tr>
</Table>
</Table>
</div>
</div>
 +
Result.<Br>
 +
<IMG Src="https://static.igem.org/mediawiki/2013/b/b5/TMU-Tokyo_0922_1.jpg"><Br>
 +
1-9: Fragment 3 - ApCm<Br>
 +
<Br>
 +
Check PCR.<Br>
<div style="text-align:center;">
<div style="text-align:center;">
<Table cellpadding="5">
<Table cellpadding="5">
<Tr>
<Tr>
-
<Td>Fragment</Td><Td>hbiF</Td><Td>Tc</Td><Td>lacI</Td><Td>FRT-Cm</Td><Td>FRT-Ap</Td>
+
<Td>Fragment</Td><Td>(control)</Td><Td>Fragment 3 - ApCm</Td><Td>Fragment 3 - ApCm</Td>
</Tr>
</Tr>
<Tr>
<Tr>
-
<Td>Primer</Td><Td>pBAD-HbiF<Br>RTcN-hbiF</Td><Td>TcN<Br>TcC3</Td><Td>141-70<Br>FRT-lacIC</Td><Td>R6Kori-Xho-3R<Br>R(FRT)2</Td><Td>lacI-R(FRT)2<Br>mhpA-SaApR</Td>
+
<Td>Primer</Td><Td>2 sets of the others</Td><Td>141-1<Br>141-20</Td><Td>141-1<Br>141-74</Td>
</Tr>
</Tr>
<Tr>
<Tr>
-
<Td>Templete</Td><Td>hbiF<Br>(BBa_K880000)</Td><Td><i>Escherichia coli</i><Br>MG1655 recA⁻<Br>(cell suspention)</Td><Td><i>Escherichia coli</i><Br>MG1655 genome</Td><Td>pSG76C</Td><Td>pSG76A2</Td>
+
<Td>Templete</Td><Td><i>Escherichia coli</i><Br>MG1655 red<Br>(Cell suspention)</Td><Td>(Cell suspention)</Td>
</Tr>
</Tr>
</Table>
</Table>
</div>
</div>
 +
Result.<Br>
 +
<IMG Src="https://static.igem.org/mediawiki/2013/9/90/TMU-Tokyo_0922_2.jpg"><Br>
 +
1-7: 141-1 and 141-20, 8: control(141-1 and 141-20),<Br>
 +
9-15: 141-1 and 141-74, 16: control(141-1 and 141-74),<Br>
 +
<Br>
 +
<Br>
 +
 +
Digestion following plasmid.<Br>
 +
<Table><Tr><Td>pSB1C3-Km-Blunt</Td></Tr></Table>
 +
<Br>
 +
<Br>
 +
P1 Preparation following fragment.<Br>
 +
<Table><Tr><Td>Fragment 1, 2</Td></Tr></Table>
 +
<Br>
 +
<Br>
 +
<b>September 23th</b><Br>
 +
PCR following fragment.
 +
<div style="text-align:center;">
 +
<Table cellpadding="5">
 +
<Tr>
 +
<Td>Fragment</Td><Td>Fragment 3 - ApCm</Td>
 +
</Tr>
 +
<Tr>
 +
<Td>Primer</Td><Td>mhpA-CmC6<Br>SaApL</Td>
 +
</Tr>
 +
<Tr>
 +
<Td>Templete</Td><Td>Fragment 3 - Cm<Br>Ap(SaApR-SaApL)</Td>
 +
</Tr>
 +
</Table>
 +
</div>
 +
Result.<Br>
 +
<IMG Src="https://static.igem.org/mediawiki/2013/c/cf/TMU-Tokyo_0923.jpg"><Br>
 +
1: Fragment 3 - Cm, 2: Fragment 3 - ApCm,<Br>
 +
3: pSB1C3-Km-Blunt<Br>
 +
<Br>
 +
<Br>
 +
Check PCR.<Br>
 +
<div style="text-align:center;">
 +
<Table cellpadding="5">
 +
<Tr>
 +
<Td>Fragment</Td><Td>Fragment 3 - Ap<Br>(control)</Td><Td>Fragment 3 - ApCm</Td>
 +
</Tr>
 +
<Tr>
 +
<Td>Primer</Td><Td>CmNR-Not<Br>SaApL</Td><Td>CmNR-Not<Br>SaApL</Td>
 +
</Tr>
 +
<Tr>
 +
<Td>Templete</Td><Td>(Cell suspention)</Td><Td>(Cell suspention)</Td>
 +
</Tr>
 +
</Table>
 +
</div>
 +
Result.<Br>
 +
<IMG Src="https://static.igem.org/mediawiki/2013/7/7a/TMU-Tokyo_0923_1.jpg">
 +
<IMG Src="https://static.igem.org/mediawiki/2013/9/94/TMU-Tokyo_0923_2.jpg"><Br>
 +
1-31: Fragment 3 - ApCm<Br>
 +
<Br>
 +
<Br>
 +
Electro Pration<Br>
 +
<Table><Tr><Td>Fragment 3 - Cm to Escherichia coli MG1655 red.</Td></Tr></Table>
 +
<Br>
 +
<Br>
 +
P1 Preparation<Br>
 +
<Table><Tr><Td>Fragment 3 - ApCm</Td></Tr></Table>
 +
<Br>
 +
<Br>
-
Result<Br>
 
-
<IMG Src="https://static.igem.org/mediawiki/2013/f/f3/TMU-Tokyo_0902.jpg" width="350" hight="305"><Br>
 
-
1: PterR1, 2: PtetR2, 3: FLP, 4: Sm, 5: araC-P,<Br>
 
-
6: hbiF, 7: Tc, 8: LacI, 9: FRT-Cm, 10: FRT-Ap<Br>
 
 +
<b>September 24th</b><Br>
 +
P1 transdunction<Br>
 +
<Table><Tr><Td>Fragment 1, 2</Td></Tr></Table>
 +
Result.<Br>
 +
<IMG Src="https://static.igem.org/mediawiki/2013/1/19/TMU-Tokyo_0924.jpg"><Br>
<Br>
<Br>
<Br>
<Br>
-
<b>September 3rd</b><Br>
 
-
Vector Remodeling with PCR. Insertion Recognition sites (Blunt).<Br>
 
<div style="text-align:center;">
<div style="text-align:center;">
<Table cellpadding="5">
<Table cellpadding="5">
<Tr>
<Tr>
-
<Td>Fragment</Td><Td>Km-Blunt</Td>
+
<Td>Fragment</Td><Td>Fragment 3 - Ap<Br>(control)</Td><Td>Fragment 3 - ApCm</Td>
</Tr>
</Tr>
<Tr>
<Tr>
-
<Td>Primer</Td><Td>Km-Blunt-N<Br>Km-Blunt-C</Td>
+
<Td>Primer</Td><Td>CmNR-Not<Br>141-1</Td><Td>CmNR-Not<Br>141-1</Td>
</Tr>
</Tr>
<Tr>
<Tr>
-
<Td>Templete</Td><Td>pACYC177</Td>
+
<Td>Templete</Td><Td>(Cell suspention)</Td><Td>(Cell suspention)</Td>
</Tr>
</Tr>
</Table>
</Table>
</div>
</div>
-
 
+
Result.<Br>
-
Result<Br>
+
<IMG Src="https://static.igem.org/mediawiki/2013/b/bc/TMU-Tokyo_0924_1.jpg"><Br>
-
<IMG Src="https://static.igem.org/mediawiki/2013/1/19/TMU-Tokyo_0903.jpg" width="144" hight="250"><Br>
+
1-16: Fragment 3 - ApCm<Br>
<Br>
<Br>
<Br>
<Br>
-
DNA Existract following fragment from gel.<Br>
+
P1 transduction<Br>
-
<Table>
+
<Table><Tr><Td>Fragment 2 to Fragment 1</Td></Tr></Table>
-
<Tr><Td>PtetR1, FLP, Sm, araC-P, hbiF, Tc, lacI, FRT-Cm, FRT-Ap, Km-Blunt</Td></Tr>
+
<Table><Tr><Td>Fragment 1 to Fragment 2</Td></Tr></Table>
-
</Table>
+
Result.<Br>
 +
<IMG Src="https://static.igem.org/mediawiki/2013/5/56/TMU-Tokyo_0924_2.jpg"><Br>
 +
<IMG Src="https://static.igem.org/mediawiki/2013/b/b4/TMU-Tokyo_0924_3.jpg" width="250" Height="125"><Br>
 +
Get Fragment 1-2<Br>
 +
<Br>
 +
Electro Poration<Br>
 +
<Table><Tr><Td>Fragment 1, 2, 3 - Ap, 3 - Cm, 3 - ApCm (in pSBC3-Km) to <i>Escherichia coli<i> DH1</Td></Tr></Table>
 +
Result.<Br>
 +
<IMG Src="https://static.igem.org/mediawiki/2013/e/eb/TMU-Tokyo_0924_4.jpg"><Br>
 +
 
 +
 
<Br>
<Br>
<Br>
<Br>
-
<b>September 4th</b><Br>
 
-
PCR following fragment. (2nd PCR)<Br>
 
 +
<b>September 25th</b><Br>
 +
Check PCR.<Br>
<div style="text-align:center;">
<div style="text-align:center;">
<Table cellpadding="5">
<Table cellpadding="5">
<Tr>
<Tr>
-
<Td>Fragment</Td><Td>Fragment 1</Td><Td>Fragment 2</Td><Td>Fragment 3</Td>
+
<Td>Fragment</Td><Td>Fragment 3 - ApCm<Br>(control)</Td><Td>Fragment 3 - ApCm</Td>
</Tr>
</Tr>
<Tr>
<Tr>
-
<Td>Primer</Td><Td>lacA-IRR/IRL<Br>cynR-SaApL</Td><Td>araCHC<Br>araA-TcC</Td><Td>R(SaApR)-lacIN<Br>mhpA-CmC6</Td>
+
<Td>Primer</Td><Td>141-49<Br>141-1</Td><Td>CmNR-Not<Br>141-1</Td>
</Tr>
</Tr>
<Tr>
<Tr>
-
<Td>Templete</Td><Td>PtetR<Br>FLP<Br>Sm</Td><Td>araC-P<Br>hbiF<Br>Tc</Td><Td>lacI<Br>FRT-Cm</Td>
+
<Td>Templete</Td><Td>(Cell suspention)</Td><Td>(Cell suspention)</Td>
</Tr>
</Tr>
</Table>
</Table>
</div>
</div>
-
 
+
Result.<Br>
-
Result<Br>
+
<IMG Src="https://static.igem.org/mediawiki/2013/8/8b/TMU-Tokyo_0925.jpg"><Br>
-
<IMG Src="https://static.igem.org/mediawiki/2013/d/d1/TMU-Tokyo_0904.jpg"><Br>
+
1-7: 141-49 and 141-1, 8: control(141-49 and 141-8),<Br>
-
1: Fragment 1, 2: Fragment 2, 3: Fragment 3<Br>
+
9-15: 141-1 and CmNR-Not, 16: control(141-1 and CmNR-Not)<Br>
<Br>
<Br>
<Br>
<Br>
-
<b>September 5th</b><Br>
+
 
-
PCR following fragment. (2nd PCR)<Br>
+
<b>September 26th</b><Br>
 +
P1 transduction<Br>
 +
<Table><Tr><Td>Fragment 3 to Fragment 1-2</Td></Tr></Table>
 +
Get Fragment 1-2-3.<Br>
 +
<Br>
 +
<Br>
 +
Check PCR<Br>
<div style="text-align:center;">
<div style="text-align:center;">
<Table cellpadding="5">
<Table cellpadding="5">
<Tr>
<Tr>
-
<Td>Fragment</Td><Td>Fragment 1-A</Td><Td>Fragment 1-B</Td><Td>Fragment 2-C</Td><Td>Fragment 2-D</Td>
+
<Td>Fragment</Td><Td>Fragment 1-2-3</Td><Td>Fragment 1-2<Br>(control)</Td>
</Tr>
</Tr>
<Tr>
<Tr>
-
<Td>Primer</Td><Td>CmNR-Stu<Br>FLP-R</Td><Td>FLP-L<Br>CmC</Td><Td>araC2<Br>RTcN-hbiF</Td><Td>pBAD-hbiF<Br>TcC3</Td>
+
<Td>Primer</Td><Td>CmNR-Not<Br>141-1</Td><Td>141-49<Br>141-1</Td>
</Tr>
</Tr>
<Tr>
<Tr>
-
<Td>Templete</Td><Td>PtetR<Br>FLP</Td><Td>FLP<Br>Sm</Td><Td>araC-P<Br>hbiF</Td><Td>hbiF<Br>Tc</Td>
+
<Td>Templete</Td><Td>(Cell suspention)</Td><Td>(Cell suspention)</Td>
</Tr>
</Tr>
</Table>
</Table>
</div>
</div>
-
Result<Br>
+
Result.<Br>
-
<IMG Src="https://static.igem.org/mediawiki/2013/9/9c/TMU-Tokyo_0905.jpg"><Br>
+
<IMG Src="https://static.igem.org/mediawiki/2013/8/8a/TMU-Tokyo_0926.jpg"><Br>
-
1: Fragment 1-A, 2: Fragment 1-B,<Br>
+
1-7: Fragment 1-2-3, 8: cotrol, 9-10: Fragment 1,<Br>
-
3: Fragment 2-C, 4: Fragment 2-D<Br>
+
11-13: Fragment 2, 14-15: Fragment 3 - Ap, 16-17: Fragment 3 - Cm<Br>
 +
18-19: Fragment 3 - AmCm<Br>
<Br>
<Br>
<Br>
<Br>
-
Check following gel extract product.<Br>
+
<b>September 27th</b><Br>
 +
Assay<Br>
 +
<PCR CHECK preculture>
 +
      Pythagorean devices
<Table>
<Table>
-
<Tr><Td>PtetR1, FLP, Sm, araC-P, hbiF, Tc, lacI, FRT-Cm, FRT-Ap, Km-Blunt</Td></Tr>
+
<Tr><Td>LB 2ml + arabinose -10%  0.1ml</Td><Td> + with the inoculating loop 1655 ins①~③ No.1</Td></Tr>
 +
<Tr><Td>LB 2ml + arabinose -10%  0.1ml </Td><Td>+ with the inoculating loop 1655 ins①~③ No.2</Td></Tr>
 +
<Tr><Td>LB 2ml + arabinose -10%  0.1ml</Td><Td> + with the inoculating loop 1655 ins①~③ No.3</Td></Tr>
 +
<Tr><Td>LB 2ml + arabinose -10%  0.1ml </Td><Td>+                    1655</Td></Tr>
 +
<Tr><Td> LB 2ml                        </Td><Td>+ with the inoculating loop 1655 ins①~③ No.1</Td></Tr>
 +
<Tr><Td>LB 2ml                        </Td><Td> + with the inoculating loop 1655 ins①~③ No.2</Td></Tr>
 +
<Tr><Td>LB 2ml                        </Td><Td>+ with the inoculating loop 1655 ins①~③ No.3</Td></Tr>
 +
<Tr><Td>LB 2ml                        </Td><Td> +                    1655</Td></Tr>
</Table>
</Table>
-
Result<Br>
+
                    ⇒I culture it at 39 degrees for 2h.
 +
<Br>
 +
<Br>
 +
<PCR CHECK Pythagorean devices FRT>
 +
<Table>
 +
 
 +
<Tr><Td>Premix(1)</Td> <Td>Cell suspension</Td> <Td>ins①-③</Td> ara(+) No.1~3</Td></Tr>
 +
<Tr><Td></Td> <Td></Td> <Td>Ins①-③</Td> ara(-) No.1~3</Td></Tr>
 +
<Tr><Td></Td> <Td></Td> <Td>1655</Td> ara(+)</Td></Tr>
 +
<Tr><Td></Td> <Td></Td> <Td>1655</Td> ara(-)</Td></Tr>
 +
<Tr><Td></Td> <Td>GXL Buff</Td> <Td>20</Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td>dNTP</Td> <Td>8</Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td>GXL</Td> <Td>2</Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td></Td> <Td> (30) </Td> <Td></Td></Tr>
 +
<Tr><Td>Premix(2) </Td> <Td>Premix(1) </Td> <Td>27</Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td>CmNR-not2</Td> <Td>9</Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td>141-1</Td> <Td>9</Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td></Td> <Td> (45) </Td> <Td></Td></Tr>
 +
<Tr><Td>mix</Td> <Td>Cell suspension</Td> <Td>5λ</Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td>Premix(2) </Td> <Td>5λ</Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td></Td> <Td><4P3-3></Td> <Td></Td></Tr>
 +
</Table>
 +
<Br>
 +
<Br>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<PCR CHECK Ptet inversion>
 +
<Table>
 +
<Tr><Td>Cell suspension</Td> <Td>1655</Td> <Td>ins①~③</Td> <Td>ara(+)</Td> No.1~3</Td></Tr>
 +
<Tr><Td></Td> <Td>1655</Td> <Td>ins①~③</Td> <Td>ara(-)</Td> No.1~3</Td></Tr>
 +
<Tr><Td>Premix①</Td> <Td>GXL Buffer</Td> <Td>30</Td> <Td></Td> <Td></Td></Tr>
 +
<Tr><Td></Td>< <Td>dNTP</Td> <Td>12</Td> <Td></Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td>GXL</Td> <Td>3</Td> <Td></Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td></Td> <Td> (45) </Td> <Td></Td> <Td></Td></Tr>
 +
<Tr><Td>Premix②</Td> <Td></Td> <Ptet1></Td> <Ptet2></Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td>Premix(1) </Td> <Td>21</Td> <Td>21</Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td>FimE-C2-EcoRI</Td> <Td>7</Td> <Td>7</Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td>TetP1</Td> <Td>7</Td> <Td></Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td>TetP2</Td> <Td></Td> <Td>7</Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td></Td> <Td> (35) </Td> <Td> (35) </Td> <Td></Td></Tr>
 +
<Tr><Td>mix</Td> <Td>Premix②</Td> <Td>5</Td> <Td></Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td>Cell suspension</Td> <Td>5</Td> <Td></Td> <Td></Td></Tr>
 +
<Tr><Td></Td> <Td><4P3-3></Td> <Td></Td> <Td></Td> <Td></Td></Tr>
 +
</Table>
 +
<Br>
 +
<Br>
 +
<plating assay Pythagorean devices>
 +
<Table>
 +
<Tr><Td>LB + arabinose (0.2%)</Td><Td>→</Td><Td>plating (single colony isolation)</Td></Tr>
 +
<Tr><Td>                  </Td><Td>→</Td><Td>1655 + ins①~③    No.1~3</Td></Tr>
 +
<Tr><Td>                  </Td><Td>→</Td><Td>1655              No.1~3</Td></Tr>
 +
<Tr><Td>  LB + IPTG (0.4g/ml) </Td><Td>→</Td><Td> plating (single colony isolation) </Td></Tr>
 +
<Tr><Td>                    </Td><Td>→</Td><Td>1655 + ins①~③    No.1~3</Td></Tr>
 +
<Tr><Td>                      </Td><Td>→</Td><Td>1655              No.1~3</Td></Tr>
 +
<Tr><Td> LB                </Td><Td>→</Td><Td>plating (single colony isolation) </Td></Tr>
 +
<Tr><Td>                </Td><Td>→</Td><Td>1655 + ins①~③    No.1~3</Td></Tr>
 +
<Tr><Td>                  </Td><Td>→</Td><Td>1655              No.1~3</Td></Tr>
 +
</Table>
 +
 
 +
 

Latest revision as of 01:25, 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
LB 2ml + arabinose -10% 0.1ml + with the inoculating loop 1655 ins①~③ No.1
LB 2ml + arabinose -10% 0.1ml + with the inoculating loop 1655 ins①~③ No.2
LB 2ml + arabinose -10% 0.1ml + with the inoculating loop 1655 ins①~③ No.3
LB 2ml + arabinose -10% 0.1ml + 1655
LB 2ml + with the inoculating loop 1655 ins①~③ No.1
LB 2ml + with the inoculating loop 1655 ins①~③ No.2
LB 2ml + with the inoculating loop 1655 ins①~③ No.3
LB 2ml + 1655
⇒I culture it at 39 degrees for 2h.

ara(+) No.1~3 ara(-) No.1~3 ara(+) ara(-)
Premix(1) Cell suspension ins①-③
Ins①-③
1655
1655
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>


No.1~3 No.1~3<
Cell suspension 1655 ins①~③ ara(+)
1655 ins①~③ ara(-)
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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