Team:NCTU Formosa/notes

From 2013.igem.org

(Difference between revisions)
Line 771: Line 771:
<li class="green e2"><p>Mini-prep of cultivated PompC , Ar  E.coli</p></li>
<li class="green e2"><p>Mini-prep of cultivated PompC , Ar  E.coli</p></li>
</ul>
</ul>
-
</div>
+
</div>
-
 
+
</div>
</div>
<div class="day">
<div class="day">
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<div class="daysmonth">
<div class="daysmonth">
<!---------------------------------------- week start ---------------------------------------->
<!---------------------------------------- week start ---------------------------------------->
-
<div class="week">
+
<div class="week bigwk">
<div class="day">
<div class="day">
-
<div class="daybar"><p>1</p></div>
+
<div class="daybar"><p></p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
-
<li class="caldot green"></li>
 
</ul>
</ul>
</div>
</div>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green"><p>PCR + single colony isolation of PompC+B003+LacI+J61048 Cr E.coli&TetR+B0015 Cr E.coli&PompC+B0032 Kr E.coli, then Electrophoresis of  these parts</p></li>
 
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>2</p></div>
+
<div class="daybar"><p>1</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
</ul>
</ul>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green"><p>DNA sequencing OK</p></li>
+
<li class="green e5"><p>Ligation: insert [B0030]ES+[ TetR+B0015]XP/vector [PSB1C3]EP </p></li>
 +
<li class="green e5"><p>transformation of this part and cultivation of this part on LB-C plate</p></li>
 +
<li class="green e5"><p>Digestion : mRFP [EP]</p></li>
 +
<li class="green e5"><p>Ligation : insert mRFP [EP]/vector pSB1C3 [EP]</p></li>
 +
<li class="green e5"><p>Transformation of mRFP ,and cultivation on LB-C plate</p></li>
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>3</p></div>
+
<div class="daybar"><p>2</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
</ul>
</ul>
</div>
</div>
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<div class="open">
<div class="open">
<ul>
<ul>
 +
<li class="green e2"><p>PCR +single colony isolation B0030+TetR+B0015  Cr E.coli</p></li>
 +
<li class="green e2"><p>Single colony isolation from mRFP LB-C plate, and cultivation of in liquid LB-C</p></li>
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>4</p></div>
+
<div class="daybar"><p>3</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
</ul>
</ul>
</div>
</div>
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<div class="open">
<div class="open">
<ul>
<ul>
 +
<li class="green e7"><p>Electrophoresis of B0030+TetR+B0015 not OK</p></li>
 +
<li class="green e7"><p>electrophoresis  of B0030+TetR+B0015  again OK</p></li>
 +
<li class="green e7"><p>Ligation: Pcons+B0030+pcyA+B0032+ho1</p></li>
 +
<li class="green e7"><p>Transformation of Pcons+B0030+pcyA+B0032+ho1</p></li>
 +
<li class="green e7"><p>Mini-prep of cultivated 37℃RBS+luxR+37℃RBS+mGFP and mRFP E.coli</p></li>
 +
<li class="green e7"><p>Digestion : mRFP [ES] & pSB1A3 [EP] & pSB1C3 [EP] & pSB1K3 [EP]</p></li>
 +
<li class="green e7"><p>Electrophoresis of insert fragment [mRFP & pSB1A3 & pSB1C3 & pSB1K3]---- OK</p></li>
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>5</p></div>
+
<div class="daybar"><p>4</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
</ul>
</ul>
</div>
</div>
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<div class="open">
<div class="open">
<ul>
<ul>
 +
<li class="green e5"><p>Cultivation of B0030+TetR+B0015+PSB1C3 in liquid LB-C tubes. PCR  of  Pcons+B0030+pcyA+B0032+ho1</p></li>
 +
<li class="green e5"><p>Electrophoresis of Pcons+B0030+pcyA+B0032+ho1</p></li>
 +
<li class="green e5"><p>Cultivation of Pcons+B0030+pcyA+B0032+ho1 with liquid LB-A</p></li>
 +
<li class="green e5"><p>Ligation : insert mRFP [ES] & J61048 [XP]/ vector pSB1K3 [EP]</p></li>
 +
<li class="green e5"><p>Transformation of mRFP+J61048 ,and cultivation on LB-K plate</p></li>
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>6</p></div>
+
<div class="daybar"><p>5</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
</ul>
</ul>
</div>
</div>
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<div class="open">
<div class="open">
<ul>
<ul>
 +
<li class="green e6"><p>Mini-prep of cultivated B0030+TetR+B0015+PSB1C3 Cr E.coli and then digest with XbaI and Pst1</p></li>
 +
<li class="green e6"><p>Electrophoresis  of[ B0030+TetR+B0015]mini&dig XP
 +
</p></li>
 +
<li class="green e6"><p>Decide to determine the sequence of B0030+TetR+B0015</p></li>
 +
<li class="green e6"><p>Digestion: [Pcons+B0030+pcyA+B0032+ho1]ES</p></li>
 +
<li class="green e6"><p>Electrophoresis of Pcons+B0030+pcyA+B0032+ho1──NOT OK</p></li>
 +
<li class="green e6"><p>PCR of insert fragment [mRFP+J61048]</p></li>
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day brn">
<div class="day brn">
-
<div class="daybar"><p>7</p></div>
+
<div class="daybar"><p>6</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
</ul>
</ul>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green"><p>digestion : pLac+B0034+zif268+AlsS (pm) [ES] &amp; B0034+ PBSII+ilvC (pm) [XP]</p></li>
+
<li class="green e5"><p>Ligation :insert [PompC]ES+[B0032]XP/vector [PSB1C3] EP and insert [LacI+J61048]ES&[Plac]XP/vector [PSB1C3]EP</p></li>
 +
<li class="green e5"><p>Transformation of PompC+B0030+PSB1C3 and LacI+J61048+Plac+PSB1C3</p></li>
 +
<li class="green e5"><p>Ligation : insert 37℃RBS+luxR+37℃RBS+mGFP [ES] & B0015 [XP]/ vector pSB1C3 [EP]</p></li>
 +
<li class="green e5"><p>Transformation of 37℃RBS+luxR+37℃RBS+mGFP+B0015 ,and cultivation on LB-C plate</p></li>
 +
<li class="green e5"><p>Electrophoresis of insert fragment [mRFP+J61048]----undetermined</p></li>
</ul>
</ul>
</div>
</div>
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<div class="week mweek">
<div class="week mweek">
<div class="day">
<div class="day">
-
<div class="daybar"><p>8</p></div>
+
<div class="daybar"><p>7</p></div>
<div class="dots">
<div class="dots">
 +
<ul>
 +
</ul>
</div>
</div>
<div class="open">
<div class="open">
 +
<ul>
 +
</ul>
</div>
</div>
</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>9</p></div>
+
<div class="daybar"><p>8</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
-
<li class="caldot green"></li>
 
-
<li class="caldot green"></li>
 
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e4"><p>Testing the temperature of the point mutation between of HivC &amp; ilvD by the m.p 50℃ of PCR</p></li>
+
<li class="green e2"><p>PCR + single colony isolation of LacI+J61048+Plac +PSB1C3 E.coli</p></li>
-
<li class="green e4"><p>digestion : pSB1K3 [EP]</p></li>
+
<li class="green e2"><p>Electrophoresis of LacI+J61048+Plac OK</p></li>
-
<li class="green e4"><p>ligation : insert pLac+B0034+zif268+AlsS (pm) [ES] &amp; B0034+ PBSII+ilvC (pm) [XP]/vector pSB1K3 [EP]</p></li>
+
-
<li class="green e4"><p>transformation of pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) and cultivation on LB-K plate</p></li>
+
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>10</p></div>
+
<div class="daybar"><p>9</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
-
<li class="caldot green"></li>
 
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e4"><p>Testing the temperature of the point mutation between of HivC &amp; ilvD by the m.p 48℃ of PCR</p></li>
+
<li class="green e3"><p>Single colony isolation from mRFP+J61048 LB-K plate, and cultivation of in liquid LB-K</p></li>
-
<li class="green e4"><p>Digestion : B0034+ HivC+ilvD+37℃ RBS [DPn1] </p></li>
+
<li class="green e3"><p>Mini-prep of cultivated mRFP+J61048 E.coli</p></li>
-
<li class="green e4"><p>PCR of insert fragment [pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC] (pm) OK</p></li>
+
<li class="green e3"><p>Digestion : mRFP+J61048 [XP]</p></li>
-
<li class="green e4"><p>SCI from pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) LB-K plate, and cultivation in liquid LB-K</p></li>
+
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>11</p></div>
+
<div class="daybar"><p>10</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
-
<li class="caldot green"></li>
 
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e3"><p>mini-prep of cultivated pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm)  E. coli </p></li>
+
<li class="green e2"><p>Cultivation
-
<li class="green e3"><p>DNA sequencing OK</p></li>
+
of LacI+J61048+Plac in liquid LB-C tubes.</p></li>
-
<li class="green e3"><p>culture condition test: activation DH5αovernight</p></li>
+
<li class="green e2"><p>Electrophoresis of insert fragment [mRFP+J61048]----NOT OK</p></li>
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>12</p></div>
+
<div class="daybar"><p>11</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
-
<li class="caldot green"></li>
 
<li class="caldot green"></li>
<li class="caldot green"></li>
</ul>
</ul>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e2"><p>transfer to new medium(1/100), OD0.2 start counting culture time</p></li>
+
<li class="green"><p>Mini-prep of cultivated LacI+J61048+Plac E.coli</p></li>
-
<li class="green e2"><p>transfer to 30゜C and 27゜C</p></li>
+
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>13</p></div>
+
<div class="daybar"><p>12</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
</ul>
</ul>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green"><p>transfer to 30゜C and 27゜C</p></li>
+
<li class="green e7"><p>Ligation again:Insert [PompC]ES&[B0032]XP/vector [PSB1K3]EP</p></li>
 +
<li class="green e7"><p>Digestion: [LacI+J61048+Plac]XP</p></li>
 +
<li class="green e7"><p>Electrophoresis of [LacI+J61048+Plac]mini&dig XP OK</p></li>
 +
<li class="green e7"><p>Decide to determine the sequence of LacI+J61048+Plac</p></li>
 +
<li class="green e7"><p>Transformation of PompC(Ar)+B0032(Ar)+PSB1K3 again but there is still no colony</p></li>
 +
<li class="green e7"><p>Transformation of 37℃RBS+luxR+37℃RBS+mGFP+B0015, and cultivation on LB-C plate</p></li>
 +
<li class="green e7"><p>Transformation of mRFP+J61048 ,and cultivation on LB-K plate.</p></li>
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day brn">
<div class="day brn">
-
<div class="daybar"><p>14</p></div>
+
<div class="daybar"><p>13</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
</ul>
</ul>
</div>
</div>
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<div class="open">
<div class="open">
<ul>
<ul>
 +
<li class="green"><p>Ligation: Insert [PompC]ES&[B0032]XP/vector [PSB1K3]EP. PCR of insert fragment [37℃RBS+luxR+37℃RBS+mGFP+B0015 & mRFP+J61048].</p></li>
</ul>
</ul>
</div>
</div>
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<div class="week mweek">
<div class="week mweek">
<div class="day">
<div class="day">
-
<div class="daybar"><p>15</p></div>
+
<div class="daybar"><p>14</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
</ul>
</ul>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green"><p>transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A plate</p></li>
+
<li class="green e3"><p>Transformation of the ligation product made yesterday on LB-K plate  but there is no colony appearing</p></li>
 +
<li class="green e3"><p>Electrophoresis of insert fragment [37℃RBS+luxR+37℃RBS+mGFP+B0015]----OK</p></li>
 +
<li class="green e3"><p>Electrophoresis of insert fragment [mRFP+J61048]----NOT OK
 +
RBS+mGFP+B0015 LB-C plate, and cultivation of in liquid LB-C. PCR of insert fragment [mRFP+J61048]----NOT OK</p></li>
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>16</p></div>
+
<div class="daybar"><p>15</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
</ul>
</ul>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green"><p>PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm)-----NOT OK</p></li>
+
<li class="green e8"><p>Ligation and trans formation of insert [ B0030]ES+ [TetR+B0015]XP/vector [PSB1C3]EP  and cultivation on LB-C plate</p></li>
 +
<li class="green e8"><p>Transformation of PSB1K3 for testing</p></li>
 +
<li class="green e8"><p>Transformation of  Pcons</p></li>
 +
<li class="green e8"><p>Digestion: [B0030+pcyA]ES</p></li>
 +
<li class="green e8"><p>Electrophoresis of Pcons──OK</p></li>
 +
<li class="green e8"><p>Electrophoresis of insert fragment [mRFP+J61048]----NOT OK</p></li>
 +
<li class="green e8"><p>Mini-prep of cultivated 37℃RBS+luxR+37℃RBS+mGFP+B0015 E.coli----NOT OK</p></li>
 +
<li class="green e8"><p>PCR of insert fragment [mRFP+J61048]----OK</p></li>
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>17</p></div>
+
<div class="daybar"><p>16</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
Line 2,894: Line 2,982:
<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e2"><p>Point mutation by PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS]</p></li>
+
<li class="green e8"><p>Digestion:[PompC]ES,[PSB1K3]&[PSB1C3]EPand Electrophoresis for checking these parts OK</p></li>
-
<li class="green e2"><p>Digestion: B0034+ HivC+ilvD+37℃ RBS [DPn1] </p></li>
+
<li class="green e8"><p>PCR + singles colony isolation of B0030+TetR+B0015 +PSB1C3 E.coli and electrophoresis of this insert fragment not OK Because the PCR electrophoresis result is strange，we conduct this experience again OK</p></li>
 +
<li class="green e8"><p>Ligation: Pcons&Pcons+B0030+pcyA+B0032+ho1&pSB1K3.</p></li>
 +
<li class="green e8"><p>Transformation of Pcons+B0032+pcyA+B0030+ho1.</p></li>
 +
<li class="green e8"><p>Cultivation of Pcons with liquid LB-C</p></li>
 +
<li class="green e8"><p>Electrophoresis of Pcons──NOT OK</p></li>
 +
<li class="green e8"><p>Single colony isolation from 37℃RBS+luxR+37℃RBS+mGFP+B0015 LB-C plate, and cultivation of in liquid LB-C</p></li>
 +
<li class="green e8"><p>Single colony isolation from mRFP+J61048 LB-K plate, and cultivation of in liquid LB-K</p></li>
</ul>
</ul>
</div>
</div>
Line 2,901: Line 2,995:
</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>18</p></div>
+
<div class="daybar"><p>17</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
</ul>
</ul>
</div>
</div>
Line 2,909: Line 3,011:
<div class="open">
<div class="open">
<ul>
<ul>
 +
<li class="green e8"><p>Transformation of PompC+B0032+PSB1C3 and
 +
Cultivation of B0030+TetR+B0015 Cr E.coli  in  liquid LB-C tube. </p></li>
 +
<li class="green e8"><p>Transformation of Pcons</p></li>
 +
<li class="green e8"><p>transformation of tetR plasmid and cultivation on LB –A plate  3. Digestion: ter. [J61048], ter. [B0015] dig EP</p></li>
 +
<li class="green e8"><p>Digestion : pSB1C3 [ES] & pSB1C3 [XP]</p></li>
 +
<li class="green e8"><p>Ligation : insert sRNA+target 1 [ES]/vector pSB1C3 [ES]</p></li>
 +
<li class="green e8"><p>Transformation of 37℃RBS+luxR+37℃RBS+mGFP+B0015 & sRNA 1 & sRNA 2 & target 1 & target 2 ,and cultivation on LB-C plate</p></li>
 +
<li class="green e8"><p>Transformation of mRFP+J61048 & sRNA+target 1 & sRNA+target 2 ,and cultivation on LB-K plate</p></li>
 +
<li class="green e8"><p>Electrophoresis of insert fragment [37℃RBS+luxR+37℃RBS+mGFP+B0015 & mRFP+J61048 & pSB1A3 & pSB1C3 & pSB1K3]-- mRFP+J61048 NOT OK Digestion : mRFP+J61048 [XP]</p></li>
</ul>
</ul>
</div>
</div>
Line 2,914: Line 3,025:
</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>19</p></div>
+
<div class="daybar"><p>18</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
</ul>
</ul>
Line 2,923: Line 3,041:
<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green"><p>transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A,K,C plate (A sucessful)</p></li>
+
<li class="green e8"><p>Mini-prep of cultivated B0030+TetR+B0015 Cr E.coli and then digest these mini with XbaI and PstI</p></li>
 +
<li class="green e8"><p>PCR + single colony isolation of PompC+B0032 Cr E.coli and electrophoresis of this  insert fragment OK Cultivation of [PompC+B0032] Cr  E.coli in liquid LB-C Tubes.</p></li>
 +
<li class="green e8"><p>Cultivation of Pcons with liquid LB-C</p></li>
 +
<li class="green e8"><p>Digestion : pSB1C3 [ES] & pSB1C3 [XP]</p></li>
 +
<li class="green e8"><p>Mini-prep of Pcons. Electrophoresis of Pcons──NOT OK</p></li>
 +
<li class="green e8"><p>Four colonies isolation from Pcons LB plate and use streak plate method to isolate the pure cultures. Digestion : mRFP+J61048 [XP]</p></li>
 +
<li class="green e8"><p>Single colony isolation from sRNA+target 1 & sRNA+target 2 LB-K plate, and cultivation of in liquid LB-K</p></li>
 +
<li class="green e8"><p>Single colony isolation from Plux LB-A plate, and cultivation of in liquid LB-A----NOT OK</p></li>
</ul>
</ul>
</div>
</div>
Line 2,929: Line 3,054:
</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>20</p></div>
+
<div class="daybar"><p>19</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
Line 2,939: Line 3,070:
<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e2"><p>PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm) ---- OK</p></li>
+
<li class="green e8"><p>Mini-prep of cultivated PompC+B0032 Cr E.coli, And then digest with EcoR1 and SpeI</p></li>
-
<li class="green e2"><p>Single colony isolation from B0034+ HivC+ilvD+37℃ RBS (pm) LB-A plate, and cultivation in liquid LB-A</p></li>
+
<li class="green e8"><p>Electrophoresis of PSB1K3 made at 7/16 and B0030+TetR+B0015, PompC+B0032 OK.</p></li>
 +
<li class="green e8"><p>Cultivation of Pcons with liquid LB-C</p></li>
 +
<li class="green e8"><p>Single colony isolation from37℃RBS+luxR+37℃RBS+mGFP+B0015 LB-C plate, and cultivation of in liquid LB-C</p></li>
 +
<li class="green e8"><p>Single colony isolation from mRFP+J61048 LB-K plate, and cultivation of in liquid LB-K(?)</p></li>
 +
<li class="green e8"><p>PCR of insert fragment [sRNA 1 & sRNA 2 & target 1 & target 2]</p></li>
 +
<li class="green e8"><p>Electrophoresis of insert fragment [sRNA 1 & sRNA 2 & target 1 & target 2]---- target 1?</p></li>
 +
<li class="green e8"><p>Mini-prep of cultivated sRNA+target 1 & sRNA+target 2 E.coli.</p></li>
</ul>
</ul>
</div>
</div>
Line 2,946: Line 3,083:
</div>
</div>
<div class="day brn">
<div class="day brn">
-
<div class="daybar"><p>21</p></div>
+
<div class="daybar"><p>20</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
Line 2,957: Line 3,099:
<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e3"><p>mini-prep of cultivated B0034+ HivC+ilvD+37℃ RBS (pm) E. coli </p></li>
+
<li class="green e8"><p>Mini-prep of Pcons.
-
<li class="green e3"><p>Digestion: pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm)(G1) [ES] &amp; B0034+ HivC+ilvD+37℃ RBS (pm)(G2) [XP] </p></li>
+
-Digestion: [Pcons]ES.</p></li>
-
<li class="green e3"><p>ligation : insert G1[ES] &amp; G2[XP]
+
<li class="green e8"><p>Electrophoresis of Pcons──NOT OK.</p></li>
-
          vector pSB1C3[EP]
+
<li class="green e8"><p>Single colony isolation from sRNA 1 & sRNA 2 & target 2 LB-C plate, and cultivation of in liquid LB-C</p></li>
-
</p></li>
+
<li class="green e8"><p>Single colony isolation from mRFP+J61048 LB-K plate, and cultivation of in liquid LB-K</p></li>
 +
<li class="green e8"><p>Transformation of Plux ,and cultivation on LB-A plate</p></li>
 +
<li class="green e8"><p>Single colony isolation from Plux & PLac & Pcons LB-A plate, and cultivation of in liquid LB-A</p></li>
 +
<li class="green e8"><p>Transformation of RBS+lacI+B0010+B0012+pLac ,and cultivation on LB-K plate</p></li>
 +
<li class="green e8"><p>Mini-prep of cultivated 37℃RBS+luxR+37℃RBS+mGFP+B0015 & mRFP+J61048 & sRNA 1 & sRNA 2 & target 2 E.coli</p></li>
</ul>
</ul>
</div>
</div>
Line 2,969: Line 3,115:
<!---------------------------------------- week end ---------------------------------------->
<!---------------------------------------- week end ---------------------------------------->
<!---------------------------------------- week start ---------------------------------------->
<!---------------------------------------- week start ---------------------------------------->
-
<div class="week">
+
<div class="week bigwk">
<div class="day">
<div class="day">
-
<div class="daybar"><p>22</p></div>
+
<div class="daybar"><p>21</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
</ul>
</ul>
Line 2,980: Line 3,130:
<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green"><p>transformation of G1+G2,and cultivation on LB- C plate failed</p></li>
+
<li class="green e5"><p>Single colony isolation from 7/15 Pcons LB plate(Red one).</p></li>
 +
<li class="green e5"><p>Cultivation of Pcons with LB-C. PCR of insert fragment [target 1]</p></li>
 +
<li class="green e5"><p>Transformation of RBS+lacI+B0010+B0012+pLac ,and cultivation on LB-K plate</p></li>
 +
<li class="green e5"><p>Electrophoresis of insert fragment [37℃RBS+luxR+37℃RBS+mGFP+B0015 & mRFP+J61048 & sRNA 1 & sRNA 2 & target 1 & target 2]----?</p></li>
 +
<li class="green e5"><p>Mini-prep of cultivated Plux & PLac E.coli
 +
Digestion : Plux [ES] & Plac [ES] & mRFP+J61048 [XP].</p></li>
</ul>
</ul>
</div>
</div>
Line 2,986: Line 3,141:
</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>23</p></div>
+
<div class="daybar"><p>22</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
</ul>
</ul>
Line 2,995: Line 3,157:
<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green"><p>ligation : insert G1[ES] &amp; G2[XP]/vector pSB1C3[EP]</p></li>
+
<li class="green e8"><p>Ligation :insert [LacI+J61048]ES&[Plac]XP/vector [PSB1C3] EP</p></li>
 +
<li class="green e8"><p>Transformation of the ligation product made at this morning and cultivation on LB-C plate.</p></li>
 +
<li class="green e8"><p>Mini-prep of Pcons+mRFP</p></li>
 +
<li class="green e8"><p>Digestion: [Pcons]ES.</p></li>
 +
<li class="green e8"><p>Electrophoresis of Pcons+mRFP──NOT OK.</p></li>
 +
<li class="green e8"><p>Cultivation of Pcons+mRFP with liquid LB-C. PCR of Pcons+mRFP</p></li>
 +
<li class="green e8"><p>Electrophoresis of Pcons+mRFP──OK.</p></li>
 +
<li class="green e8"><p>Electrophoresis of insert fragment [Plux & Plac & mRFP+J61048?]----?</p></li>
</ul>
</ul>
</div>
</div>
Line 3,001: Line 3,170:
</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>24</p></div>
+
<div class="daybar"><p>23</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
Line 3,012: Line 3,186:
<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e3"><p>transformation of G1+G2,and cultivation on LB- C plate</p></li>
+
<li class="green e8"><p>PCR +Single colony isolation  of   LacI+J61048+Plac and electrophoresis Of  this  insert fragment  OK</p></li>
-
<li class="green e3"><p>Digestion: G2’(B0034+HIVC+ilvD) [XP]</p></li>
+
<li class="green e8"><p>Mini-prep of Pcons-mRFP.</p></li>
-
<li class="green e3"><p>ligation : insert G1[ES] &amp; G2‘[XP]/vector pSB1C3[EP]</p></li>
+
<li class="green e8"><p>Digestion: [B0030]XP&[B0032]XP&[B0023]XP&[Pcons]ES</p></li>
 +
<li class="green e8"><p>Electrophoresis of B0030,B0032,B0034,Pcons──OK</p></li>
 +
<li class="green e8"><p>Transformation of B0030+pcyA+B0032+ho1+B0030/B0032/B0034</p></li>
 +
<li class="green e8"><p>Electrophoresis of insert fragment [mRFP+J61048]----?</p></li>
 +
<li class="green e8"><p>Transformation of RBS+lacI+B0010+B0012+Plac ,and cultivation on LB-K plate----NOT OK</p></li>
 +
<li class="green e8"><p>Single colony isolation from RBS+lacI+B0010+B0012+pLac & mRFP+J61048 LB-K plate, and cultivation of in liquid LB-K</p></li>
</ul>
</ul>
</div>
</div>
Line 3,020: Line 3,199:
</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>25</p></div>
+
<div class="daybar"><p>24</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
Line 3,030: Line 3,215:
<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e2"><p>PCR of insert fragment [kivD+B0015]</p></li>
+
<li class="green e8"><p>Cultivation of LacI+J61048+Plac E.coli in liquid LB-C tubes</p></li>
-
<li class="green e2"><p>transformation of G1+G2’, and cultivation on LB- C plate failed</p></li>
+
<li class="green e8"><p>Mini-prep of cultivated LacI+J61048+Plac E.coli. </p></li>
 +
<li class="green e8"><p>Cultivation of B0030+pcyA+B0032+ho1+B0032 with liquid LB</p></li>
 +
<li class="green e8"><p>Transformation of B0030+pcyA+B0032+ho1+B0030/B0032/B0034</p></li>
 +
<li class="green e8"><p>Mini-prep of cultivated mRFP+J61048 E.coli</p></li>
 +
<li class="green e8"><p>Digestion : mRFP+J61048 [XP]</p></li>
 +
<li class="green e8"><p>Transformation of target 1 ,and cultivation on LB-C plate</p></li>
 +
<li class="green e8"><p>Single colony isolation from RBS+lacI+B0010+B0012+pLac plate, and cultivation of in liquid LB-without antibiotics----NO E.coli?</p></li>
</ul>
</ul>
</div>
</div>
Line 3,037: Line 3,228:
</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>26</p></div>
+
<div class="daybar"><p>25</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
Line 3,047: Line 3,244:
<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e2"><p>Single colony isolation from kivD+B0015 LB-C plate, and cultivation in liquid LB-C</p></li>
+
<li class="green e8"><p>Digestion of LacI+J61048+Plac Cr [XP]</p></li>
-
<li class="green e2"><p>sample and do GC</p></li>
+
<li class="green e8"><p>Electrophoresis of[ LacI+J61048 +Plac]dig XP  OK</p></li>
 +
<li class="green e8"><p>Decide to determine the sequence of  LacI+J61048 +Plac</p></li>
 +
<li class="green e8"><p>Ligation :insert [B0030]ES& [TetR+B0015]XP/vector [PSB1K3]EP.</p></li>
 +
<li class="green e8"><p>Cultivation of B0030+pcyA+B0032+ho1+B0030/B0032/B0034 with liquid LB.</p></li>
 +
<li class="green e8"><p>PCR of insert fragment [target 1]</p></li>
 +
<li class="green e8"><p>Single colony isolation from RBS+lacI+B0010+B0012+pLac plate , and cultivation of in liquid LB-without antibiotics----NO E.coli?</p></li>
 +
<li class="green e8"><p>Electrophoresis of insert fragment [mRFP+J61048 & target 1]----?</p></li>
</ul>
</ul>
</div>
</div>
Line 3,054: Line 3,257:
</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>27</p></div>
+
<div class="daybar"><p>26</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
</ul>
</ul>
Line 3,063: Line 3,272:
<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green"><p>mini-prep of cultivated kivD+B0015 E. coli </p></li>
+
<li class="green e7"><p>Transformation of B0030+TetR+B0015+PSB1K3 and cultivation on LB-K plate. -Mini-prep of B0030+pcyAB0032+ho1+B0030/B0032/B0034.</p></li>
 +
<li class="green e7"><p>Digestion: [B0030+pcyA+B0032+ho1+B0030/B0032/B0034]XP</p></li>
 +
<li class="green e7"><p>Electrophoresis of digested B0030+pcyA+B0032+ho1+B0030/B0032/B0034──OK</p></li>
 +
<li class="green e7"><p>Electrophoresis of B0030,B0032,B0034,Pcons──OK</p></li>
 +
<li class="green e7"><p>Mini-prep of cultivated target 1 & mRFP+J61048 E.coli</p></li>
 +
<li class="green e7"><p>Digestion: target 1 [ES] & mRFP [XP]?</p></li>
 +
<li class="green e7"><p>Electrophoresis of insert fragment [RBS+lacI+B0010+B0012+pLac & mRFP & target 1]----?</p></li>
</ul>
</ul>
</div>
</div>
Line 3,069: Line 3,284:
</div>
</div>
<div class="day brn">
<div class="day brn">
-
<div class="daybar"><p>28</p></div>
+
<div class="daybar"><p>27</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
</ul>
</ul>
</div>
</div>
Line 3,077: Line 3,295:
<div class="open">
<div class="open">
<ul>
<ul>
 +
<li class="green e3"><p>PCR and single colony isolation of B0030+TetR+B0015 Kr E.coli</p></li>
 +
<li class="green e3"><p>Cultivation of B0030+TetR+B0015 in liquid LB-tube.</p></li>
 +
<li class="green e3"><p>Ligation: pSB1C3&Pcons&B0030+pcyA+B0032+ho1+B0030</p></li>
</ul>
</ul>
</div>
</div>
Line 3,084: Line 3,305:
<!---------------------------------------- week end ---------------------------------------->
<!---------------------------------------- week end ---------------------------------------->
<!---------------------------------------- week start ---------------------------------------->
<!---------------------------------------- week start ---------------------------------------->
-
<div class="week">
+
<div class="week mweek">
<div class="day">
<div class="day">
-
<div class="daybar"><p>29</p></div>
+
<div class="daybar"><p>28</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
Line 3,096: Line 3,319:
<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e2"><p>Point mutation by PCR of insert fragment [B0034+ HivC+ilvD(G2’)]</p></li>
+
<li class="green e4"><p>Cultivation of B0030+TetR+B0015 in liquid LB-K tubes again</p></li>
-
<li class="green e2"><p>Digestion: G2’ [DPn1]</p></li>
+
<li class="green e4"><p>Ligation:insert[PompC+B0032]ES+[LacI+J61048+Plac]XP/vector[PSB1K3]EP</p></li>
 +
<li class="green e4"><p>Mini-prep of cultivated B0030+TetR+B0015 Kr E.coli</p></li>
 +
<li class="green e4"><p>Transformation of Pcons+B0030+pcyA+B0032+ho1+B0030</p></li>
</ul>
</ul>
</div>
</div>
Line 3,103: Line 3,328:
</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>30</p></div>
+
<div class="daybar"><p>29</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
</ul>
</ul>
Line 3,112: Line 3,338:
<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green"><p>transformation of G2’, and cultivation on LB- A plate</p></li>
+
<li class="green e2"><p>Transformation of PompC+B0032+LacI+J61048+Plac+PSB1K3. -Transformation of Cph8.</p></li>
 +
<li class="green e2"><p>Digestion: [Pcons]XP.</p></li>
</ul>
</ul>
</div>
</div>
Line 3,118: Line 3,345:
</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>31</p></div>
+
<div class="daybar"><p>30</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
Line 3,128: Line 3,356:
<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e2"><p>Digestion: kivD+B0015 [EP] (checking bp----failed)</p></li>
+
<li class="green e3"><p>Ligation:insert[PompC+B0032]ES+[LacI+J61048+Plac]XP/vector[PSB1K3]EP&[PSB1A3]EP</p></li>
-
<li class="green e2"><p>Single colony isolation from G2’ (pm) LB-A plate, and cultivation in liquid LB-A</p></li>
+
<li class="green e3"><p>Transformation of PompC+B0032+LacI+J61048+Plac+PSB1K3&PSB1A3.</p></li>
 +
<li class="green e3"><p>Electrophoresis of Pcons ─OK. Digestion: [Pcons]SP.</p></li>
</ul>
</ul>
</div>
</div>
Line 3,135: Line 3,364:
</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p></p></div>
+
<div class="daybar"><p>31</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
</ul>
</ul>
</div>
</div>
Line 3,143: Line 3,376:
<div class="open">
<div class="open">
<ul>
<ul>
 +
<li class="green e4"><p>PCR + single colony isolation of PompC+B0032+LacI+J61048+Plac+PSB1K3&PSB1A3 E.coli</p></li>
 +
<li class="green e4"><p>Electrophoresis of PompC+B0032+LacI+J61048+Plac OK.</p></li>
 +
<li class="green e4"><p>Ligation: Pcons&B0030+pcyA+B0032+ho1+B0030.</p></li>
 +
<li class="green e4"><p>Transformation of Pcons+B0030+pcyA+B0032+ho1+B0030</p></li>
</ul>
</ul>
</div>
</div>

Revision as of 11:07, 21 September 2013

Notes

The experimental log that records down the purpose, method, result and date of each experiment that we have conducted.

About the notes

Our notes are the abbreviations of the experimental process, including titles, genes we transformed, plasmids we used, and resistances on the plasmids. We categorized these notes by date. Moreover, team members followed the iGEM protocol in each experimental step in order to get better experimental efficiency and the results we had expected.

March 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

24

25

26

27

28

29

  • Transformation of ho1(Kr), Cph8(Cr), 32℃ RBS(Ar) and cultivation on three LB plates.

  • transformation of PompC and cultivation on LB-A plate

30

  • Single colony isolation from three plates and cultivation them in liquid LB

  • Single colony isolation from PompC LB-C plate and cultivation in liquid LB-A

  • mini-prep of cultivated PompC E.coli

31

  • Transformation of RBS B0030+PSB1A3&B0034+PSB1A3 and cultivation on LB-A plate.

  • Mini-prep for cultivated ho1(Kr), Cph8(Cr), 32℃ RBS(Ar) PCR of ho1(Kr), Cph8(Cr), 32℃ RBS(Ar).

  • Electrophoresis of ho1(Kr), Cph8(Cr), 32℃ RBS(Ar) ──NOT OK.

  • PCR of insert fragment [PompC+psB1A2]

April 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

  • Cultivation of B0030&B0034 Ar E.coli in liquid LB-A tubes.

2

  • Mini-prep of cultivated B0030&B0034 E.coli and then digestion: [B0030]XP&[B0034]XP.

3

4

5

6

  • Electrophoresis of [B0030]XP&[B0034]XP Not OK.

  • PCR of ho1 to check ho1 and transform to test the resistance.

7

  • Electrophoresis of [B0030]mini&dig XP and [B0034]mini&dig XP OK

  • Cultivation of B0030&B0034 Ar E.coli in liquid LB-A tubes.

  • Cultivation of ho1 in liquid LB-K and LB-K plate

  • Cultivation of Cph8+RBS in liquid LB-C.

    • 8

    • Mini-prep of cultivated B0030&B0034 Ar E.coli. Mini-prep of ho1 & Cph8+RBS.

    9

    • Electrophoresis of mini ho1&Cph8+RBS.

    10

    11

    • PCR of mini ho1&Cph8+RBS

    • Electrophoresis of ho1&Cph8+RBS(After PCR)

    • Digestion: [ho1]EP&[Cph8+RBS]EP.

    12

    • Transformation of Plac&Ptet& pcyA but there is no colony appearing in pcyA plate.

    • Electrophoresis of digested ho1&Cph8+RBS (NOT OK).

    13

    14

    • Cultivation of Ptet&Plac E.coli in LB tubes.

    • Transformation: LuxR,B0015,J61048 and cultivation on LB-A plate

    15

    • Mini-prep of cultivated Ptet&Plac E.coli

    • Digestion:[Ptet]ES&[Plac]ES PCR of insert fragment pcyA

    • Electrophoresis of [Ptet]dig ES&[pcyA]PCR&[Plac]dig ES.

    • Single colony isolation from J61048,B0015,LuxR plate and cultivation in liquid LB-A mini-prep of cultivated LuxR,J61048,B0015

    16

    • PCR of mini Cph8+RBS

    • Electrophoresis of Cph8+RBS(After PCR)──NOT OK.

    • digestion: [J61048]EP,[B0015]EP,[LuxR]EP

    • Electrophoresis of ter. mini [J61048] dig E/EP and t er. mini [B0015] dig E/EP and luxR mini dig E/EP

    17

    • PCR of mini Cph8+RBS Electrophoresis of Cph8+RBS(After PCR)──NOT OK.

    • Electrophoresis of the products of digestion of ter. [J61048], ter. [B0015] , luxR

    • transformation of tetR plasmid and cultivation on LB –A plate 3. Digestion: ter. [J61048], ter. [B0015] dig EP

    18

    19

    • Cultivation of pcyA Kr E.coli on LB-K plates

    • mini-prep of cultivated tetR E.coli

    • digestion: tetR+pSB1A2 dig EP

    • electrophoresis of tetR dig EP,ter.[J61048 ] dig EP and ter.[B0015] dig EP

    20

    • Cultivation of pcyA Kr E.coli in liquid LB-K tubes.

    • PCR of tetR,ter.[J61048] and ter.[B0015 ]mini

    • electrophoresis of PCR products of tetR mini,J61048 mini and B0015 mini

    21

    • Single colony isolation of pcyA Kr E.coli

    • Mini-prep of cultivated pcyA Kr E.coli

    • Digestion:[pcyA]ES

    • Electrophoresis of [pcyA]mini&dig ES OK

    • PCR of tetR,ter.[J61048] and ter.[B0015 ]mini

    • electrophoresis of PCR products of tetR mini,J61048 mini and B0015 mini

    • electrophoresis of the digestion products of tetR dig EP and B0015 dig EP

    • aliquot every 20ul of PompC,J61048 and LuxR

    22

    23

    24

    25

    26

    27

    28

    • Transformation of PompC mini and cultivation on LB-A plate

    29

    • transformation of PompC , Ar mini and cultivation on LB-A plate

    • single colony isolation from PompC , Ar

    30

    • Transformation of 37ﾟC RBS and ho1.

    • Mini-prep of cultivated PompC , Ar E.coli

    May 2013

    Sunday

    Monday

    Tuesday

    Wednesday

    Thursday

    Friday

    Saturday

    1

    • Cultivation of 37ﾟC RBS and ho1

    • Digestion: [pSB1C3] EP&[B0030]ES&{pcyA]XP

    • Transformation of pSB1A3 &pSB1K3

    • Transformation of mGFP[pSB1A2] and cultivation on LB-A plate.

    2

    • Cultivation of PompC&B0034&LacI&Plac&B0030&TetR&J61048&B0015 Ar E.coli in liquid LB-A tubes.

    • Cultivation of pSB1A3&pSB1K3 with liquid LB

    • Mini-prep of 37ﾟC RBS and ho1──Fail

    • Ligation: [pSB1C3] EP&[B0030]ES& {pcyA]XP

    • Transformation of B0030

    • Cultivation of 37ﾟC RBS and ho1 with liquid LB

    • Transformation of RBS+pcyA+psB1C3.

    • Transformation of mGFP[pSB1A2] and cultivation on LB-A plate

    3

    • Mini-prep of cultivated PompC &B0034&LacI&Plac&B0030&TetR&J61048&B0015,and then digestion:[PompC ]ES&[B0034]XP&[LacI]ES&[Plac]XP&[B0030]ES&[TetR]ES&[TetR]XP&[J61048]XP&[J61048]ES&[B0015]XP.

    • Mini-prep of ho1,pSB1A3, pSB1K3 and 37ﾟC RBS

    • Digestion: [B0030+pcyA]XP

    • Cultivation of B0030 with liquid LB-C

    • Check RBS+pcyA+psB1C3(PCR & Electrophoresis)──FAIL

    • Digestion: [Pcons]ES& [ho1]XP.

    • Single colony isolation from 37。C RBS Ar,pSB1A3,pSB1C3 and pSB1K3

    • electrophoresis of mGFP dig E

    4

    • Electrophoresis of [PompC]ES&[B0030]ES&[LacI]ES&[TetR]ES&[J61048]XP&[TetR]ES&[B0015]XP OK

    • Ligation: insert [PompC]ES+[B0030]XP&[LacI]ES+[J61048]XP&[J61048]ES+[Plac]XP&[B0030]ES+[TetR]XP&[TetR]ES+[B0015]XP/vector[PSB1C3]EP

    • Transformation of B0030+ho1.

    • Mini-prep of cultivated 37。C RBS Ar pSB1A3,37。C RBS+mGFP, Kr and pSB1K3 E.coli

    • digestion:37。C RBS dig ES,mGFP dig XP,LuxR dig XP and pSB1K3 dig EP

    • electrophoresis of digestion products of LuxR,37。C RBS, mGFP and pSB1K3

    • ligation:37。C RBS+luxR+pSB1K3

    • transformation of 37。C RBS+mGFP, Kr->pSB1K3 and cultivation on LB-K plate

    5

    • Electrophoresis of digested RBS+pcyA──OK

    • Ligation: RBS+ho1

    • Cultivation of J61048 with liquid LB-C

    • Digestion: [pSB1A3]EP &[pSB1K3]EP

    • Transformation of ligated RBS+ho1.

    • Ligation:37。C RBS+luxR+pSB1K3 and 37。C RBS+mGFP+pSB1K3

    • transformation of 37。C RBS+luxR, Kr and 37。C RBS+mGFP ,Kr and cultivation on LB-K plate

    6

    • Mini-prep of J61048

    • Ligation: RBS+pcyA+Pcons&pSB1K3

    • Electrophoresis of J61048.

    • Digestion: [J61048]XP

    • Transformation of RBS+pcyA+Pcons.

    • Check LB-A&C&K plates.

    7

    • Mini-prep of J61048 -Ligation: RBS+pcyA+Pcons&pSB1K3

    • Electrophoresis of J61048.

    • Digestion: [J61048]XP

    • Transformation of RBS+pcyA+Pcons.-Check LB-A&C&K plates.

    8

    • Ligation:insert[PompC]ES+[B0034]XP&[LacI]ES+[J61048]XP&[B0030]ES+[TetR]XP&[TetR]ES+[B0015]XP/vector[PSB1C3]E

    • PCR of 37。RBS+luxR, Kr and 37。C RBS+mGFP ,Kr

    • electrophoresis of 37。RBS+luxR ,Kr and 37。C RBS+mGFP ,Kr

    9

    • Transformation and cultivation of PompC+B0034&LacI+J61048&B0030+TetR&TetR+B0015(PSB1C3) on LB-C plates.

    • PCR of 37。RBS+luxR, Kr and 37。C RBS+mGFP ,Kr

    • electrophoresis of 37。RBS+luxR, Kr and 37。C RBS+mGFP ,Kr

    • single colony isolation from37。RBS+luxR, Kr and 37。C RBS+mGFP ,Kr

    10

    • Cultivation of Pcons+RBS+pcyA and B0030 with liquid LB.

    • Mini-prep of cultivated 37。C RBS+luxR, Kr E.coli and 37。C RBS+mGFP ,Kr E.coli

    • digestion: 37。RBS+luxR, dig ES and 37。C RBS+mGFP dig ES

    11

    • Mini-prep of Pcons+RBS+pcyA.

    • Electrophoresis of Pcons+RBS+pcyA──NOT OK.

    • Electrophoresis of 37。C RBS+mGFP, Kr mini, 37。C RBS+mGFP dig ES, 37。C RBS+luxR, Kr mini and 37。C RBS+luxR dig ES

    12

    • Electrophoresis of 37。C+luxR mini, dig ES and 37。C RBS+mGFP mini,dig ES

    13

    • Cultivation of TetR+B0015 Cr E.coli in liquid LB-C tube

    • Single colony isolation of backbone PSB1K3 E.coli

    • Ligation:insert[B0030]ES+[TetR]XP&[TetR]ES+[B0015]XP/vector[PSB1C3]EP.

    • Electrophoresis of Pcons+RBS+pcyA──NOT OK

    • Transformation of B0032&B0034.

    14

    • Ligation:insert[PompC]ES+[B0034]XP&[LacI]ES+[J61048]XP/vector[PSB1C3]EP.

    • Cultivation of Pcons+RBS+pcyA & B0032&B0034 with liquid LB.

    15

    • Mini-prep of cultivated PSB1K3 E.coli

    • Cultivation of B0015+TetR+PSB1C3 and TetR+B0015+PSB1C3 in liquid LB-C plates and Plac+PSB1A3 in liquid LB-A plate. Mini-prep of Pcons+RBS+pcyA&B0032&B0034.

    16

    • Ligation:insert[PompC]ES+[B0034]XP&[LacI]ES+[J61048]XP/vector[PSB1K3]EP

    • Transformation and cultivation of PompC+B0034&LacI+J61048 (PSB1K3) on LB-K plates.

    17

    • cultivation of PompC+B0034&LacI+J61048 Kr E.coli on liquid LB-K tubes.

    • single colony isolation of PompC+B0034&LacI+J61048 Kr E.coli.

    • Electrophoresis of Pcons+RBS+pcyA(NOT OK) &B0032&B0034

    • Cultivation of Pcons+RBS+pcyA.Digestion: [B0032]ES &[B0023]ES.

    18

    • Cultivation of PompC+B0034&LacI+J61048 Kr E.coli from single colony isolation plate made yesterday

    • Min-prep of cultivated [B0030+TetR]Cr&[ Plac]Ar&[PompC+B0034]Kr&[LacI+J61048]Kr E.coli and then digestion:[PompC+B0034]ES &[LacI+J61048]XP. Mini-prep Pcons+RBS+pcyA

    • Electrophoresis of Pcons+RBS+pcyA& digested B0032&B0034──NOT OK

    • Digestion: [B0032]ES &[B0034]ES &[Pcons+RBS+pcyA]E.

    19

    • Electrophoresis of [LacI+J61048]mini & dig XP and [PompC+B0034]mini & dig ES and [PSB1K3]mini & dig EP.

    • Electrophoresis of digested B0032&B0034&Pcons+RBS+pcyA──OK

    • Transformation of pSB1C3

    20

    • Digestion:[Plac]P.

    • Ligation: Pcons+RBS+pcyA&pSB1K3, B0032+ho1&pSB1C3, B0034+ho1&pSB1C1

    • Transformation of Pcons+RBS+pcyA&pSB1K3, B0032+ho1&pSB1C3, B0034+ho1&pSB1C1

    • Single colony isolation from pSB1C3

    21

    • Single colony isolation of TetR+B0015 Cr Ecoli

    • Electrophoresis of [PompC+B0034]mini&dig ES and[Plac]mini&dig P.

    • Cultivation of Pcons+RBS+pcyA &B0032+ho1 with liquid LB

    • Mini-prep of cultivated pSB1C3 E.coli

    22

    • Digestion:[Plac]XP

    • Electrophoresis of [PompC+B0034]mini&dig ES and[Plac]mini&dig XP

    • Ligation: insert[TetR]ES+[B]XP/vector[PSBC3]EP

    • Mini-prep of Pcons+RBS+pcyA

    • Mini-prep of cultivated 37。RBS+mGFP E.coli

    • digestion:37。C RBS+mGFP dig ES and mini

    • electrophoresis of 37。C RBS+mGFP mini and 37。C RBS+mGFP dig ES.

    23

    • Single colony isolation of TetR+B0015 transformation of TetR +B0015 +PSB1C3 on LB-C plates

    • Ligation +transformation on: insert[TetR]ES+[B0015]XP&[PompC]ES+[B0034]/vector[PSB1K3]EP on LB-K plates

    24

    • Single colony isolation of PompC+B0034+PSB1K3 E.coli

    • Electrophoresis of Pcons+RBS+pcyA.

    • -Ligation: B0032+ho1&B0034+ho1.

    • Digestion; [Pcons+RBS+pcyA]ES &[ho1]XP&[pSB1C3]EP.

    • Electrophoresis of 37。 RBS+mGFP dig ES

    25

    • Ligation and transformation: insert [PompC]ES+[B0034]XP/vector[PSB1K3]EP on LB-K plates

    • Single colony isolation of PompC+B0034+PSB1K3 E.coli again

    • Mini-prep of cultivated TetR+B0015 Cr E.coli and digestion:[TetR+B0015]P

    • Electrophoresis of :[TetR+B0015]P

    • Digestion: [TetR+B0015]XP

    • Cultivation of PompC+B0034 in liquid LB-K tubes.

    • Mini-prep of B0032+ho1

    • Cultivation of B0034+ho1 with liquid LB-A

    26

    • Cultivation of PompC+B0034Kr E.coli in liquid LB-K tubes again

    • Mini-prep of cultivate PompC+B0034 Kr E.coli

    • Electrophoresis of [TetR+B0015] dig XP not OK

    • Mini-prep of B0034+ho1 Electrophoresis of Pcons+RBS+pcyA & B0032+ho1 & B0034+ho1.

    27

    • Digestion:[TetR+B0015]XP

    • Electrophoresis of [TetR+B0015]mini&dig XP

    • Digestion:[PompC+B0034]E first

    • Electrophoresis of [PompC+B0034]E

    • Digestion: [PompC+B0034]E, S later

    • Ligation: insert[PompC]ES+[B0034]XP/vector[PSB1K3]EP

    28

    • Electrophoresis of [PompC+B0034]ES not OK

    • Transformation of PompC+B0034+PSB1K3.

    • Electrophoresis of B0034+ho1

    • Digestion: [B0034+ho1]XP &[Pcons+RBS+pcyA]E

    29

    • PCR and electrophoresis test. Electrophoresis of digested B0034+ho1 &Pcons+RBS+pcyA.

    30

    • Cultivation of PompC+B0034+PSB1K3 E.coli in liquid LB-K tubes

    • Ligation: insert[TetR]ES+[B0015]XP/vector[PSB1K3]EP

    • Transformation of Plac+PSB1A3 on LB-A plate

    • Digestion:B0015 Ar dig XP, pSB1A3 Ar dig EP and pSB1C3 Cr dig EP

    • ligation:37。C RBS+mGFP+ter. [B0015], Cr

    • transformation of 37。C RBS+mGFP+ter., Cr

    31

    • Mini-prep of cultivated PompC+B0034 Kr E.coli, then

    • Digestion and electrophoresis of [PompC+B0034]E

    • Decide to determine the sequence of PompC+B0015.

    • Cultivation of ho1 with liquid LB-K and pSB1C3 with liquid LB-C.

    • PCR of 37。C RBS+mGFP+ter. Cr

    • electrophoresis of 37。C RBS+mGFP+ter., ter.[B0015] digXP, pSB1C3 dig EP and pSB1A3 dig EP

    • transformation of BBa _E1010 Kr

    June 2013

    Sunday

    Monday

    Tuesday

    Wednesday

    Thursday

    Friday

    Saturday

    1

    • PCR and single colony isolation of TetR+B0015+PSB1K3

    • Electrophoresis of PCR product made at this morning and Plac(5/30 Ar) OK

    • Digestion:[Ter(B0015)]XP

    • Transformation of Ter(B0015) on LB-A plate & mGFP on LB-K plate & 37oC RBS+mGFP+Ter(B0015) on LB-C plate

    • Ligation: insert 37oC RBS[ES] & Ter(B0015)[XP]/ vector pSB1C3[EP]

    • Single colony isolation from Ter(B0015) LB-A plate, and cultivation in liquid LB-A

    2

    • Ligation: insert [TetR]ES+[B0015]XP/vector [PSB1K3]EP and transformation of this part.

    • Ligation: B0032+ho1&B0034+ho1

    • Transformation of B0032+ho1&B0034+ho1

    • Cultivation of pSB1C3(C20&C10)with liquid LB.

    • PCR of insert fragment[37oC RBS+mGFP+Ter(B0015)]

    • Transformation of mRFP on LB-A plate& LB-C plate& LB-K plate-Mini-prep of cultivated Ter(B0015) E.coli.

    3

    • PCR of Plac &TetR+B0015

    • Mini-prep of pSB1C3

    • Cultivation of B0032+ho1&B0034+ho1 with LB-A plate PCR of B0032+ho1&B0034+ho1

    • Electrophoresis of B0032+ho1&B0034+ho1──NOT OK.

    • Single colony isolation from 37oC RBS+mGFP+Ter(B0015) LB-K plate, and cultivation in liquid LB-K & mRFP LB-A, and cultivation in liquid LB-K

    • Mini-prep of cultivated 37oC RBS+mGFP+Ter(B0015) E.coli

    • Digestion:[37oC RBS+mGFP+Ter(B0015)]XP

    4

    • Electrophoresis of Plac .TetR+B0015 OK

    • Cultivation of Plac.TetR+B0015 in liquid LB-A tubes. PCR of B0032+ho1&B0034+ho1

    • Electrophoresis of B0032+ho1(OK)&B0034+ho1(After PCR)

    • Mini-prep of B0032+ho1. Digestion: [B0032+ho1]XP.

    • Cultivation of ho1 with liquid LB-K

    • Single colony isolation from mRFP LB-A plate, and cultivation in liquid LB-A

    • Digestion:[37oC RBS+mGFP+Ter(B0015)]XP

    • PCR of insert fragment[37oCRBS+mGFP+Ter(B0015)].

    5

    • Mini-prep of cultivated Plac and TetR+B0015 E.coli and then conduct digestion :[Plac]P&[TetR+B0015]P

    • Electrophoresis of [Plac ]P and [TetR+B0015]P,[Plac]XP and [TetR+B0015]XP

    • Ligation: insert[PompC+B0034]ES+[LacI+J61048]XP/vector [PSB1A3]EP

    • Electrophoresis of B0032+ho1 mini&B0032+ho1(digested)&pSB1C3 mini&pSB1C3(digested)&ho1 mini&ho1(digested)

    • Mini-prep of ho1

    • Digestion: [ho1]XP

    • Digestion:[37oC RBS+mGFP+Ter(B0015)]XP

    • Transformation of mRFP on LB-A plate

    6

    • Transformation of PompC+B0034+LacI+J61048+PSB1A3

    • Ligation: insert [PompC+B0034]ES+[LacI+J61048]XP/vector [PSB1A3]EP

    • Electrophoresis of B0032+ho1 mini&B0032+ho1(digested)&ho1 mini&ho1 (digested).

    • Single colony isolation from mRFP LB-A plate, and cultivation in liquid LB-A & LB-K & LB

    • Digestion:[Ter(B0015)]EX

    • Ligation: insert 37oC RBS+mGFP[ES]&Ter(B0015)[EX]

    • Transformation of 37oCRBS+mGFP+Ter(B0015) on LB-K plate & LB-A plate, mRFP on LB-A plate

    7

    • Transformation of PompC+B0034+LacI+J61048 again

    • Single colony isolation of TetR+B0015 Kr E.coli

    • Digestion:[LacI+J61048]ES. PCR of insert fragment[37oCRBS+mGFP+Ter(B0015)]

    • Single colony isolation from 37oCRBS+mGFP+Ter(B0015) LB-A plate, and cultivation in liquid LB-A.

    8

    • PCR + single colony isolation of PompC+B0034+LacI+J61048 E.coli

    • Electrophoresis of PompC+B0034+LacI+J61048 OK

    • Ligation: insert [LacI+J61048]ES+[Plac]XP/vector[PSB1C3]EP

    • Transformation of LacI+J61048+Plac on LB-C plate

    • Cultivation of PompC+B0034+LacI+J61048 in LB-A tubes & TetR+B0015 in LB-K tube & PSB1A3 in LB-A tube &PSB1C3 in LB-C tube.

    • Mini-prep of cultivated 37oCRBS+mGFP+Ter(B0015) E.coli

    • Digestion:[ 37oCRBS+mGFP+Ter(B0015)]XP

    • PCR of insert fragment[37oCRBS+mGFP+Ter(B0015)].

    9

    • Mini-prep from cultivated PompC+B0034+LacI+J61048 Eco.li&PSB1C3 E.coli

    • digestion : [PompC+B0034+LacI+J61048]ES&[PSB1C3]EP

    • Electrophoresis of [PompC+B0034+LacI+J61048] ES& [LacI+J61048]ES&[PSB1C3]EP.

    • Single colony isolation from 37oCRBS + mGFP+Ter(B0015)

    10

    • Mini-prep of cultivated 37oCRBS+mGFP+Ter(B0015) E.coli

    • Digestion:[ 37oCRBS+mGFP+Ter(B0015)]XP

    • Single colony isolation from 37oCRBS+mGFP+Ter(B0015) LB-A plate, and cultivation in liquid LB-A.

    11

    • PCR of insert fragment[37oCRBS+mGFP+Ter(B0015)]

    • Transformation of mRFP on LB-K plate.

    12

    • Single colony isolation from mRFP LB-K plate, and cultivation in liquid LB-K

    13

    • Ligation: insert[PompC+B0034]ES+[LacI+J61048]XP/vector[PSB1C3]EP and insert[TetR]ES+[B0015]XP/vector[PSB1C3]EP.

    • Mini-prep of cultivated mRFP E.coli

    • Digestion:[mRFP]ES & [Ter(J61048)]XP & [37OCRBS+mGFP]XP

    • Ligation: insert 37oCRBS+LuxR[ES]&37oCRBS+mGFP[XP] /vector pSB1A3[EP]

    • Transformation of 37oCRBS +LuxR+37oCRBS+mGFP on LB-A plate & mRFP +Ter(J61048) on LB-C plate& Ter(J61048) on LB-A plate.

    14

    • Transformation of PompC+B0034+LacI+J61048+PSB1C3 and TetR+B0015+PSB1C3

    • Cultivation of B0032+ho1 with liquid LB-A. PCR of insert fragment[Ter(B0015)]

    • Single colony isolation from Ter(J61048) LB-A plate, and cultivation in liquid LB-A & 37OCRBS+mGFP on LB-K plate, and cultivation in liquid LB-K

    • Transformation of 37oCRBS +LuxR+37oCRBS+mGFP on LB-A plate & mRFP +Ter(J61048) on LB-C plate

    • Mini-prep of cultivated Ter (J61048)& 37oCRBS+mGFP E.coli

    15

    • Digestion:[B0032]XP

    • Transformation of PompC+B0034+LacI+J61048 +PSB1C3and TetR+B0015 +PSB1C3 again

    • Ligation: insert[PompC]ES+[B0032]XP/vector[PSB1K3]EP, then transformation of this part

    • Mini-prep of B0032+ho1. PCR of insert fragment[37oCRBS + LuxR + 37oCRBS+mGFP]&[mRFP+Ter(J61048)].

    9

    • Mini-prep from cultivated PompC+B0034+LacI+J61048 Eco.li&PSB1C3 E.coli

    • digestion : [PompC+B0034+LacI+J61048]ES&[PSB1C3]EP

    • Electrophoresis of [PompC+B0034+LacI+J61048] ES& [LacI+J61048]ES&[PSB1C3]EP.

    • Single colony isolation from 37oCRBS + mGFP+Ter(B0015)

    10

    • Mini-prep of cultivated 37oCRBS+mGFP+Ter(B0015) E.coli

    • Digestion:[ 37oCRBS+mGFP+Ter(B0015)]XP

    • Single colony isolation from 37oCRBS+mGFP+Ter(B0015) LB-A plate, and cultivation in liquid LB-A.

    11

    • PCR of insert fragment[37oCRBS+mGFP+Ter(B0015)]

    • Transformation of mRFP on LB-K plate.

    12

    • Single colony isolation from mRFP LB-K plate, and cultivation in liquid LB-K

    13

    • Ligation: insert[PompC+B0034]ES+[LacI+J61048]XP/vector[PSB1C3]EP and insert[TetR]ES+[B0015]XP/vector[PSB1C3]EP.

    • Mini-prep of cultivated mRFP E.coli

    • Digestion:[mRFP]ES & [Ter(J61048)]XP & [37OCRBS+mGFP]XP

    • Ligation: insert 37oCRBS+LuxR[ES]&37oCRBS+mGFP[XP] /vector pSB1A3[EP]

    • Transformation of 37oCRBS +LuxR+37oCRBS+mGFP on LB-A plate & mRFP +Ter(J61048) on LB-C plate& Ter(J61048) on LB-A plate.

    14

    • Transformation of PompC+B0034+LacI+J61048+PSB1C3 and TetR+B0015+PSB1C3

    • Cultivation of B0032+ho1 with liquid LB-A. PCR of insert fragment[Ter(B0015)]

    • Single colony isolation from Ter(J61048) LB-A plate, and cultivation in liquid LB-A & 37OCRBS+mGFP on LB-K plate, and cultivation in liquid LB-K

    • Transformation of 37oCRBS +LuxR+37oCRBS+mGFP on LB-A plate & mRFP +Ter(J61048) on LB-C plate

    • Mini-prep of cultivated Ter (J61048)& 37oCRBS+mGFP E.coli

    15

    • Digestion:[B0032]XP

    • Transformation of PompC+B0034+LacI+J61048 +PSB1C3and TetR+B0015 +PSB1C3 again

    • Ligation: insert[PompC]ES+[B0032]XP/vector[PSB1K3]EP, then transformation of this part

    • Mini-prep of B0032+ho1. PCR of insert fragment[37oCRBS + LuxR + 37oCRBS+mGFP]&[mRFP+Ter(J61048)].

    16

    • PCR + single colony isolation of PompC+B003+LacI+J61048 Cr E.coli&TetR+B0015 Cr E.coli&PompC+B0032 Kr E.coli, then Electrophoresis of these parts

    17

    • Mini-prep of cultivated PompC+B0034 E.coli and TetR+B0015 Cr E.coli

    18

    19

    20

    • Ligation: Pcons+RBS+pcyA&B0032+ho1&pSB1A3.

    21

    • Ligation: insert[PompC+B0034]ES+[LacI+J61048]XP/vector[PSB1A3]EP

    • Transformation of Pcons+RBS+pcyA+B0032+ho1 on LB-A plate. PCR of insert fragment[37oCRBS + LuxR + 37oCRBS+mGFP]

    • Digestion:[pSB1C3]EP&[mRFP]ES&[Ter(J61048)]XP

    • Ligation: insert mRFP[ES]&Ter(J61048)[XP] /vector pSB1C3[EP]

    • Transformation of mRFP + Ter (J61048) on LB-C plate

    • Single colony isolation from 37oCRBS + LuxR +37oCRBS+mGFP LB-A plate, and cultivation in liquid LB-A.

    22

    • Transformation of PompC+B0034+LacI+J61048+PSB1A3 but there is no colony appearing. PCR of insert fragment[mRFP+Ter(J61048)]

    • Mini-prep of cultivated 37o CRBS + LuxR + 37oCRBS+mGFP E.coli

    • Digestion:[ 37o CRBS + LuxR + 37oCRBS+mGFP]ES

    23

    • Digestion:[PompC+B0032]ES and [TetR+B0015]XP,[LacI+J61048]XP

    • Ligation: insert [PompC+B0034]ES+[LacI+J61048]XP/vector[PSB1C3]EP

    • Transformation of PompC+B0034+LacI+J61048 +PSB1C3

    • Electrophoresis of [PompC+B0032]ES & [TetR+B0015]XP

    • Digestion again:[PompC+B0032]ES&[LacI+J61048]XP&[LacI+B0015]XP

    • Transformation of mRFP + Ter (J61048) on LB-C plate

    24

    • PCR and single colony isolation of PompC+B0034+LacI+J61048+PSB1C3 E.coli

    • Electrophoresis of [B0032]dig ES&[TetR+B0015]dig XP&[LacI+J61048]dig XP&PCR product [PompC+B0034+LacI+J61048] ,[TetR+B0015] OK

    • Decide to determine the sequence of [TetR+B0015] OK.

    • Single colony isolation from 6/21 plate.

    • Cultivation of Pcons+ RBS+pcyA+B0032+ho1 with liquid LB-A.

    • Electrophoresis of Pcons+RBS+PcyA+B0032+ho1.

    • PCR of insert fragment[mRFP+Ter(J61048)].

    25

    • Ligation:insert[PompC+B0034]ES+[LacI+J61048]XP&[PompC]ES+[B0032]XP/vector[PSB1C3]EP and [PompC+B0034]ES+[LacI+J61048]XP/vector[PSB1A3]EP

    • Transformation of the ligation product made today

    • PCR of Pcons+RBS+pcyA+B0032+ho1.

    • Cultivation of Pcons+RBS+pcyA+B0032+ho1 with liquid LB-A

    • Electrophoresis of Pcons+RBS+pcyA+B0032+ho1.

    • Mini-prep of Pcons+RBS+pcyA+B0032+ho1.

    • Ligation: insert mRFP[ES]&Ter(J61048)[XP]/vector pSB1C3[EP]

    26

    • PCR and single colony isolation of PompC+B0034+LacI+J61048+PSB1A3& PompC+B0034+LacI+J61048+PSB1C3&PompC+B0032+PSB1C3 E.coli

    • Electrophoresis of the PCR product made today

    • Electrophoresis of Pcons+RBS+pcyA+B0032+ho1──NOT OK

    • Ligation: Pcons+RBS+pcyA&B0032+ho1

    • Transformation of Pcons+RBS+pcyA+B0032+ho1. Transformation of mRFP + Ter (J61048) on LB-C plate

    27

    • Cultivation of PompC+B0034+LacI+J61048+PSB1A3 E.coli in liquid LB-A tubes

    • PCR of Pcons+RBS+pcyA+B0032+ho1

    • Cultivation of Pcons+RBS+pcyA+B0032+ho1 with liquid LB-A

    • Electrophoresis of Pcons+RBS+pcyA+B0032+ho1──NOT OK

    • Transformation of mRFP + Ter (J61048) on LB-C plate

    28

    • PCR of Pcons+RBS+pcyA+B0032+ho1

    • Electrophoresis of Pcons+RBS+pcAa+B0032+ho1──NOT OK

    • Ligation: insert mRFP[ES]&Ter(J61048)[XP] /vector pSB1C3[EP]

    29

    • Cultivation of PompC+B0034+LacI+J61048+PSB1A3 E.coli in liquid LB-A tubes again.

    • Ligation: Pcons+RBS+pcyA&B0032+ho1&pSB1A3

    • Transformation of ligated Pcons+RBS+pcyA+B0032+ho1

    • Single colony isolation from 6/21 Pcons+RBS+pcyA+B0032+ho1 LB plate

    • PCR of 6/21 Pcons+RBS+pcyA+B0032+ho1

    • Electrophoresis of 621 Pcons+RBS+pcyA+B0032+ho1─NOT OK

    • Transformation of mRFP+Ter(J61048) on LB-C plate

    • Single colony isolation from 37oCRBS +LuxR +37oCRBS+mGFP LB-A plate, and cultivation in liquid LB-A

    30

    • Mini-prep of cultivated PompC+B0034+LacI+J61048+PSB1A3 E.coli and then digest with EcoR1 and SpeI:[ PompC+B0034+LacI+J61048]dig ES

    • Electrophoresis of [PompC+B0034+LacI+J61048+PSB1A3] mini and [ PompC+B0034+LacI+J61048]dig ES OK

    • Decide to determine sequence of[ PompC+B0034+LacI+J61048]

    • Cultivation of 6/29 Pcons+RBS+PcyA+B0032+ho1 LB plate with liquid LB-A

    • PCR of 6/29 Pcons+RBS+PcyA+B0032+ho1

    • Cultivation of 6/29 re-ligated Pcons+RBS+PcyA+B0032+ho1

    • PCR of 6/29 re-ligated Pcons+RBS+pcyA+B0032+ho1

    • Electrophoresis of all above─NOT OK

    July 2013

    Sunday

    Monday

    Tuesday

    Wednesday

    Thursday

    Friday

    Saturday

    1

    • Ligation: insert [B0030]ES+[ TetR+B0015]XP/vector [PSB1C3]EP

    • transformation of this part and cultivation of this part on LB-C plate

    • Digestion : mRFP [EP]

    • Ligation : insert mRFP [EP]/vector pSB1C3 [EP]

    • Transformation of mRFP ,and cultivation on LB-C plate

    2

    • PCR +single colony isolation B0030+TetR+B0015 Cr E.coli

    • Single colony isolation from mRFP LB-C plate, and cultivation of in liquid LB-C

    3

    • Electrophoresis of B0030+TetR+B0015 not OK

    • electrophoresis of B0030+TetR+B0015 again OK

    • Ligation: Pcons+B0030+pcyA+B0032+ho1

    • Transformation of Pcons+B0030+pcyA+B0032+ho1

    • Mini-prep of cultivated 37℃RBS+luxR+37℃RBS+mGFP and mRFP E.coli

    • Digestion : mRFP [ES] & pSB1A3 [EP] & pSB1C3 [EP] & pSB1K3 [EP]

    • Electrophoresis of insert fragment [mRFP & pSB1A3 & pSB1C3 & pSB1K3]---- OK

    4

    • Cultivation of B0030+TetR+B0015+PSB1C3 in liquid LB-C tubes. PCR of Pcons+B0030+pcyA+B0032+ho1

    • Electrophoresis of Pcons+B0030+pcyA+B0032+ho1

    • Cultivation of Pcons+B0030+pcyA+B0032+ho1 with liquid LB-A

    • Ligation : insert mRFP [ES] & J61048 [XP]/ vector pSB1K3 [EP]

    • Transformation of mRFP+J61048 ,and cultivation on LB-K plate

    5

    • Mini-prep of cultivated B0030+TetR+B0015+PSB1C3 Cr E.coli and then digest with XbaI and Pst1

    • Electrophoresis of[ B0030+TetR+B0015]mini&dig XP

    • Decide to determine the sequence of B0030+TetR+B0015

    • Digestion: [Pcons+B0030+pcyA+B0032+ho1]ES

    • Electrophoresis of Pcons+B0030+pcyA+B0032+ho1──NOT OK

    • PCR of insert fragment [mRFP+J61048]

    6

    • Ligation :insert [PompC]ES+[B0032]XP/vector [PSB1C3] EP and insert [LacI+J61048]ES&[Plac]XP/vector [PSB1C3]EP

    • Transformation of PompC+B0030+PSB1C3 and LacI+J61048+Plac+PSB1C3

    • Ligation : insert 37℃RBS+luxR+37℃RBS+mGFP [ES] & B0015 [XP]/ vector pSB1C3 [EP]

    • Transformation of 37℃RBS+luxR+37℃RBS+mGFP+B0015 ,and cultivation on LB-C plate

    • Electrophoresis of insert fragment [mRFP+J61048]----undetermined

    7

    8

    • PCR + single colony isolation of LacI+J61048+Plac +PSB1C3 E.coli

    • Electrophoresis of LacI+J61048+Plac OK

    9

    • Single colony isolation from mRFP+J61048 LB-K plate, and cultivation of in liquid LB-K

    • Mini-prep of cultivated mRFP+J61048 E.coli

    • Digestion : mRFP+J61048 [XP]

    10

    • Cultivation of LacI+J61048+Plac in liquid LB-C tubes.

    • Electrophoresis of insert fragment [mRFP+J61048]----NOT OK

    11

    • Mini-prep of cultivated LacI+J61048+Plac E.coli

    12

    • Ligation again:Insert [PompC]ES&[B0032]XP/vector [PSB1K3]EP

    • Digestion: [LacI+J61048+Plac]XP

    • Electrophoresis of [LacI+J61048+Plac]mini&dig XP OK

    • Decide to determine the sequence of LacI+J61048+Plac

    • Transformation of PompC(Ar)+B0032(Ar)+PSB1K3 again but there is still no colony

    • Transformation of 37℃RBS+luxR+37℃RBS+mGFP+B0015, and cultivation on LB-C plate

    • Transformation of mRFP+J61048 ,and cultivation on LB-K plate.

    13

    • Ligation: Insert [PompC]ES&[B0032]XP/vector [PSB1K3]EP. PCR of insert fragment [37℃RBS+luxR+37℃RBS+mGFP+B0015 & mRFP+J61048].

    14

    • Transformation of the ligation product made yesterday on LB-K plate but there is no colony appearing

    • Electrophoresis of insert fragment [37℃RBS+luxR+37℃RBS+mGFP+B0015]----OK

    • Electrophoresis of insert fragment [mRFP+J61048]----NOT OK RBS+mGFP+B0015 LB-C plate, and cultivation of in liquid LB-C. PCR of insert fragment [mRFP+J61048]----NOT OK

    15

    • Ligation and trans formation of insert [ B0030]ES+ [TetR+B0015]XP/vector [PSB1C3]EP and cultivation on LB-C plate

    • Transformation of PSB1K3 for testing

    • Transformation of Pcons

    • Digestion: [B0030+pcyA]ES

    • Electrophoresis of Pcons──OK

    • Electrophoresis of insert fragment [mRFP+J61048]----NOT OK

    • Mini-prep of cultivated 37℃RBS+luxR+37℃RBS+mGFP+B0015 E.coli----NOT OK

    • PCR of insert fragment [mRFP+J61048]----OK

    16

    • Digestion:[PompC]ES,[PSB1K3]&[PSB1C3]EPand Electrophoresis for checking these parts OK

    • PCR + singles colony isolation of B0030+TetR+B0015 +PSB1C3 E.coli and electrophoresis of this insert fragment not OK Because the PCR electrophoresis result is strange，we conduct this experience again OK

    • Ligation: Pcons&Pcons+B0030+pcyA+B0032+ho1&pSB1K3.

    • Transformation of Pcons+B0032+pcyA+B0030+ho1.

    • Cultivation of Pcons with liquid LB-C

    • Electrophoresis of Pcons──NOT OK

    • Single colony isolation from 37℃RBS+luxR+37℃RBS+mGFP+B0015 LB-C plate, and cultivation of in liquid LB-C

    • Single colony isolation from mRFP+J61048 LB-K plate, and cultivation of in liquid LB-K

    17

    • Transformation of PompC+B0032+PSB1C3 and Cultivation of B0030+TetR+B0015 Cr E.coli in liquid LB-C tube.

    • Transformation of Pcons

    • transformation of tetR plasmid and cultivation on LB –A plate 3. Digestion: ter. [J61048], ter. [B0015] dig EP

    • Digestion : pSB1C3 [ES] & pSB1C3 [XP]

    • Ligation : insert sRNA+target 1 [ES]/vector pSB1C3 [ES]

    • Transformation of 37℃RBS+luxR+37℃RBS+mGFP+B0015 & sRNA 1 & sRNA 2 & target 1 & target 2 ,and cultivation on LB-C plate

    • Transformation of mRFP+J61048 & sRNA+target 1 & sRNA+target 2 ,and cultivation on LB-K plate

    • Electrophoresis of insert fragment [37℃RBS+luxR+37℃RBS+mGFP+B0015 & mRFP+J61048 & pSB1A3 & pSB1C3 & pSB1K3]-- mRFP+J61048 NOT OK Digestion : mRFP+J61048 [XP]

    18

    • Mini-prep of cultivated B0030+TetR+B0015 Cr E.coli and then digest these mini with XbaI and PstI

    • PCR + single colony isolation of PompC+B0032 Cr E.coli and electrophoresis of this insert fragment OK Cultivation of [PompC+B0032] Cr E.coli in liquid LB-C Tubes.

    • Cultivation of Pcons with liquid LB-C

    • Digestion : pSB1C3 [ES] & pSB1C3 [XP]

    • Mini-prep of Pcons. Electrophoresis of Pcons──NOT OK

    • Four colonies isolation from Pcons LB plate and use streak plate method to isolate the pure cultures. Digestion : mRFP+J61048 [XP]

    • Single colony isolation from sRNA+target 1 & sRNA+target 2 LB-K plate, and cultivation of in liquid LB-K

    • Single colony isolation from Plux LB-A plate, and cultivation of in liquid LB-A----NOT OK

    19

    • Mini-prep of cultivated PompC+B0032 Cr E.coli, And then digest with EcoR1 and SpeI

    • Electrophoresis of PSB1K3 made at 7/16 and B0030+TetR+B0015, PompC+B0032 OK.

    • Cultivation of Pcons with liquid LB-C

    • Single colony isolation from37℃RBS+luxR+37℃RBS+mGFP+B0015 LB-C plate, and cultivation of in liquid LB-C

    • Single colony isolation from mRFP+J61048 LB-K plate, and cultivation of in liquid LB-K(?)

    • PCR of insert fragment [sRNA 1 & sRNA 2 & target 1 & target 2]

    • Electrophoresis of insert fragment [sRNA 1 & sRNA 2 & target 1 & target 2]---- target 1?

    • Mini-prep of cultivated sRNA+target 1 & sRNA+target 2 E.coli.

    20

    • Mini-prep of Pcons. -Digestion: [Pcons]ES.

    • Electrophoresis of Pcons──NOT OK.

    • Single colony isolation from sRNA 1 & sRNA 2 & target 2 LB-C plate, and cultivation of in liquid LB-C

    • Single colony isolation from mRFP+J61048 LB-K plate, and cultivation of in liquid LB-K

    • Transformation of Plux ,and cultivation on LB-A plate

    • Single colony isolation from Plux & PLac & Pcons LB-A plate, and cultivation of in liquid LB-A

    • Transformation of RBS+lacI+B0010+B0012+pLac ,and cultivation on LB-K plate

    • Mini-prep of cultivated 37℃RBS+luxR+37℃RBS+mGFP+B0015 & mRFP+J61048 & sRNA 1 & sRNA 2 & target 2 E.coli

    21

    • Single colony isolation from 7/15 Pcons LB plate(Red one).

    • Cultivation of Pcons with LB-C. PCR of insert fragment [target 1]

    • Transformation of RBS+lacI+B0010+B0012+pLac ,and cultivation on LB-K plate

    • Electrophoresis of insert fragment [37℃RBS+luxR+37℃RBS+mGFP+B0015 & mRFP+J61048 & sRNA 1 & sRNA 2 & target 1 & target 2]----?

    • Mini-prep of cultivated Plux & PLac E.coli Digestion : Plux [ES] & Plac [ES] & mRFP+J61048 [XP].

    22

    • Ligation :insert [LacI+J61048]ES&[Plac]XP/vector [PSB1C3] EP

    • Transformation of the ligation product made at this morning and cultivation on LB-C plate.

    • Mini-prep of Pcons+mRFP

    • Digestion: [Pcons]ES.

    • Electrophoresis of Pcons+mRFP──NOT OK.

    • Cultivation of Pcons+mRFP with liquid LB-C. PCR of Pcons+mRFP

    • Electrophoresis of Pcons+mRFP──OK.

    • Electrophoresis of insert fragment [Plux & Plac & mRFP+J61048?]----?

    23

    • PCR +Single colony isolation of LacI+J61048+Plac and electrophoresis Of this insert fragment OK

    • Mini-prep of Pcons-mRFP.

    • Digestion: [B0030]XP&[B0032]XP&[B0023]XP&[Pcons]ES

    • Electrophoresis of B0030,B0032,B0034,Pcons──OK

    • Transformation of B0030+pcyA+B0032+ho1+B0030/B0032/B0034

    • Electrophoresis of insert fragment [mRFP+J61048]----?

    • Transformation of RBS+lacI+B0010+B0012+Plac ,and cultivation on LB-K plate----NOT OK

    • Single colony isolation from RBS+lacI+B0010+B0012+pLac & mRFP+J61048 LB-K plate, and cultivation of in liquid LB-K

    24

    • Cultivation of LacI+J61048+Plac E.coli in liquid LB-C tubes

    • Mini-prep of cultivated LacI+J61048+Plac E.coli.

    • Cultivation of B0030+pcyA+B0032+ho1+B0032 with liquid LB

    • Transformation of B0030+pcyA+B0032+ho1+B0030/B0032/B0034

    • Mini-prep of cultivated mRFP+J61048 E.coli

    • Digestion : mRFP+J61048 [XP]

    • Transformation of target 1 ,and cultivation on LB-C plate

    • Single colony isolation from RBS+lacI+B0010+B0012+pLac plate, and cultivation of in liquid LB-without antibiotics----NO E.coli?

    25

    • Digestion of LacI+J61048+Plac Cr [XP]

    • Electrophoresis of[ LacI+J61048 +Plac]dig XP OK

    • Decide to determine the sequence of LacI+J61048 +Plac

    • Ligation :insert [B0030]ES& [TetR+B0015]XP/vector [PSB1K3]EP.

    • Cultivation of B0030+pcyA+B0032+ho1+B0030/B0032/B0034 with liquid LB.

    • PCR of insert fragment [target 1]

    • Single colony isolation from RBS+lacI+B0010+B0012+pLac plate , and cultivation of in liquid LB-without antibiotics----NO E.coli?

    • Electrophoresis of insert fragment [mRFP+J61048 & target 1]----?

    26

    • Transformation of B0030+TetR+B0015+PSB1K3 and cultivation on LB-K plate. -Mini-prep of B0030+pcyAB0032+ho1+B0030/B0032/B0034.

    • Digestion: [B0030+pcyA+B0032+ho1+B0030/B0032/B0034]XP

    • Electrophoresis of digested B0030+pcyA+B0032+ho1+B0030/B0032/B0034──OK

    • Electrophoresis of B0030,B0032,B0034,Pcons──OK

    • Mini-prep of cultivated target 1 & mRFP+J61048 E.coli

    • Digestion: target 1 [ES] & mRFP [XP]?

    • Electrophoresis of insert fragment [RBS+lacI+B0010+B0012+pLac & mRFP & target 1]----?

    27

    • PCR and single colony isolation of B0030+TetR+B0015 Kr E.coli

    • Cultivation of B0030+TetR+B0015 in liquid LB-tube.

    • Ligation: pSB1C3&Pcons&B0030+pcyA+B0032+ho1+B0030

    28

    • Cultivation of B0030+TetR+B0015 in liquid LB-K tubes again

    • Ligation:insert[PompC+B0032]ES+[LacI+J61048+Plac]XP/vector[PSB1K3]EP

    • Mini-prep of cultivated B0030+TetR+B0015 Kr E.coli

    • Transformation of Pcons+B0030+pcyA+B0032+ho1+B0030

    29

    • Transformation of PompC+B0032+LacI+J61048+Plac+PSB1K3. -Transformation of Cph8.

    • Digestion: [Pcons]XP.

    30

    • Ligation:insert[PompC+B0032]ES+[LacI+J61048+Plac]XP/vector[PSB1K3]EP&[PSB1A3]EP

    • Transformation of PompC+B0032+LacI+J61048+Plac+PSB1K3&PSB1A3.

    • Electrophoresis of Pcons ─OK. Digestion: [Pcons]SP.

    31

    • PCR + single colony isolation of PompC+B0032+LacI+J61048+Plac+PSB1K3&PSB1A3 E.coli

    • Electrophoresis of PompC+B0032+LacI+J61048+Plac OK.

    • Ligation: Pcons&B0030+pcyA+B0032+ho1+B0030.

    • Transformation of Pcons+B0030+pcyA+B0032+ho1+B0030

    August 2013

    Sunday

    Monday

    Tuesday

    Wednesday

    Thursday

    Friday

    Saturday

    1

    • mini-prep of cultivated G2’ E. coli

    2

    3

    4

    5

    6

    7

    8

    9

    • Digestion: G1 [ES] & G2‘ [XP] & pSB1C3 [EP]

    • ligation : insert G1[ES] & G2‘[XP]/vector pSB1C3[EP]

    10

    • strain test: activation of different strains overnight

    11

    • transfer to new medium, OD0.2 IPTGinduction, culture in 37゜C

    12

    • Single colony isolation from G1+G2’ LB-C plate, and cultivation in liquid LB-C

    • transfer to 27゜C

    13

    • mini-prep of cultivated G1+G2’ E. coli

    14

    15

    • Digestion: Ptet+B0032[ES] & GliI+KivD[XP]

    • sample and do GC

    16

    • ligation : insert Ptet+B0032[ES] & GliI+KivD[XP]/vector pSB1K3[EP]

    • Single colony isolation from G1+G2 LB-C plate, and cultivation in liquid LB-C

    17

    • transformation of Ptet+B0032+ GliI+KivD, and cultivation on LB- K plate

    • mini-prep of cultivated G1+G2 E. coli

    18

    • PCR of insert fragment [Ptet+B0032+ GliI+KivD]-----OK

    • DNA sequencing------NOT OK

    19

    20

    21

    22

    23

    24

    • carbon source test: activation DH5α overnight

    25

    • transfer to new medium(1/100), OD0.2 start counting culture time

    26

    • culturing for 72hours, inject feeding solution per 24hours

    27

    • culturing for 72hours, inject feeding solution per 24hours

    28

    • transformation of DNA program, and cultivation on LB- A plate

    • culturing for 72hours, inject feeding solution per 24hours

    29

    • ligation : insert Ptet+B0032[ES] & GliI+KivD[XP]/vector pSB1K3[EP]

    • Single colony isolation from DNA program LB-C plate, and cultivation in liquid LB-C

    • sample & do GC

    30

    • transformation of Ptet+B0032+ GliI+KivD, and cultivation on LB- K plate

    • mini-prep of cultivated DNA program E. coli

    • Do GC

    31

    • PCR of insert fragment [Ptet+B0032+ GliI+KivD] OK

    • DNA sequencing NOT OK

    September 2013

    Sunday

    Monday

    Tuesday

    Wednesday

    Thursday

    Friday

    Saturday

    1

    • ligation : insert Zif268 [ES] & AlsS [XP]/Vector pSB1K3 [EP]

    2

    • transformation of Zif268+AlsS+pSB1K3 and cultivation on LB-K plate

    3

    4

    • digestion : [pSB1K3] EP

    • digestion : [pSB1K3] EP

    5

    6

    7

    • PCR of insert fragment [Zif268+AlsS] OK

    • DNA Sequencing OK

    • ligation : point mutation HivC

    • TA cloning : point mutation HivC

    8

    9

    • Single colony isolation from Zif268+AlsS LB-K plate, and cultivation of in liquid LB-K

    10

    • mini-prep of cultivated Zif268+ AlsS E. coli

    • transformation of point mutation HivC and cultivation on LB-A plate

    • transformation of B0034 and cultivation on LB-A plate

    11

    12

    • Single colony isolation from HivC LB-A plate, and in liquid LB-A

    • Single colony isolation from B0034 LB-A plate, and in liquid LB-A

    13

    • mini-prep of cultivated point mutation HivC E. coli & B0034 E. coli

    14

    • digestion : Zif268+AlsS [XP]

    15

    16

    17

    • Single colony isolation from ilvD LB-A plate, and cultivation in liquid LB-A

    18

    • mini-prep of cultivated ilvD E. coli

    • digestion : ilvD [EP] & pSB1K3 [EP]

    • transformation of 37℃ RBS and cultivation on LB-C plate

    19

    • Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A

    20

    • digestion : B0034 [SP]

    • gel extraction

    • ligation :insert Zif268+AlsS [XP]/Vector B0034 [SP]

    • mini-prep of cultivated Hivc E. coli

    21

    • transformation of B0034+Zif268+AlsS and cultivation on LB-A plate

    • transformation of HivC and cultivation on LB-A plate

    22

    • PCR of insert fragment [B0034+Zif268+AlsS] OK

    • DNA Sequencing NOT OK

    • digestion : B0034 [SP] & Zif268+AlsS [XP]

    • gel extraction

    • ligation :insert Zif268+AlsS [XP]/Vector B0034+pSB1A3 [SP]

    • transformation of B0034+Zif268+AlsS and cultivation on LB-A plate

    • Single colony isolation from B0034 LB-A plate, and cultivation in liquid LB-A

    • SCI from ilvD LB-K plate, and cultivation in liquid LB-K

    23

    • PCR of insert fragment [B0034+Zif268+AlsS] NOT OK

    • transformation of B0034+Zif268+AlsS and cultivation on LB-A plate

    • mini-prep of cultivated B0034 and ilvD E. coli

    • Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A

    24

    • mini-prep of cultivated HivC E. coli

    • digestion : B0034 [SP] & ilvD [XP]

    25

    • digestion : pSB1C3 [EP] & HivC [ES] & HivC [SP]

    • ligation :insert HivC [ES] & ilvD [XP]/Vector pSB1C3 [EP]

    • ligation :insert ilvD [XP]/Vector HivC+pSB1A3 [SP]

    26

    • transformation of HivC+ilvD and cultivation on LB-A & LB-C plate

    27

    • PCR of insert fragment [B0034+Zif268+AlsS] NOT OK

    28

    • transformation of pSB1A3 & pSB1C3 and cultivation on LB-A & LB-C plate

    • PCR of insert fragment [HivC+ilvD] OK

    • DNA sequencing NOT OK

    29

    • Single colony isolation from pSB1C3 LB-C plate, and cultivation in liquid LB-C

    30

    • mini-prep of cultivated & pSB1C3 E. coli

    Retrieved from "http://2013.igem.org/Team:NCTU_Formosa/notes"