Team:ZJU-China/Notebook.html

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<title>Notebook - July</title>
<title>Notebook - July</title>
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<h1>Week1</h1>
<h1>Week1</h1>
-
<table id="notesheet" width="650" border="1" cellspacing="0"
+
<table id="notesheet" border="1" cellspacing="0"
cellpadding="1" style="vertical-align: middle">
cellpadding="1" style="vertical-align: middle">
<tr>
<tr>
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cellpadding="1">
cellpadding="1">
-
<td width="128">鈥?Aerobic cultivation of DH5伪</td>
+
<td width="128">▪Aerobic cultivation of DH5α</td>
-
<td width="196">鈥?Preparation of apparatus for the formation of
+
<td width="196">▪ Preparation of apparatus for the formation of
biofilm</td>
biofilm</td>
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<table width="217" border="0" cellspacing="0" cellpadding="1">
<table width="217" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="215">鈥eceiving primers ordered previously</td>
+
<td width="215">▪eceiving primers ordered previously</td>
</tr>
</tr>
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<table width="238" border="0" cellspacing="0" cellpadding="1">
<table width="238" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="200">鈥?Preparation of the aliquot of the primers</td>
+
<td width="200">▪ Preparation of the aliquot of the primers</td>
-
<td width="200">鈥?Something wrong with a shaking incubator</td>
+
<td width="200">▪ Something wrong with a shaking incubator</td>
</tr>
</tr>
</table>
</table>
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<table width="222" border="0" cellspacing="0" cellpadding="1">
<table width="222" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="155">鈥?Preparation of culture plates for the
+
<td width="155">▪ Preparation of culture plates for the
transformations</td>
transformations</td>
</tr>
</tr>
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<table width="313" border="0" cellspacing="0" cellpadding="1">
<table width="313" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="127">鈥?Preparation of culture plates for the
+
<td width="127">▪ Preparation of culture plates for the
transformations</td>
transformations</td>
-
<td width="182">鈥?protocols of transformation</td>
+
<td width="182">▪ protocols of transformation</td>
</tr>
</tr>
</table>
</table>
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<table width="246" border="0" cellspacing="0" cellpadding="1">
<table width="246" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td>鈥?Several colonies were picked up and cultivated in 5mL LB
+
<td>▪ Several colonies were picked up and cultivated in 5mL LB
-
medium. 鈥ryosectioning of biofilm</td>
+
medium. ▪ryosectioning of biofilm</td>
</tr>
</tr>
</table>
</table>
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cellpadding="1">
cellpadding="1">
-
<td width="128">鈥?Set up new LB culture plates with ampicillin
+
<td width="128">▪ Set up new LB culture plates with ampicillin
and kanamycin
and kanamycin
</p>
</p>
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-
<td width="196">鈥?路Sterilization of Glycerol and <br />
+
<td width="196">▪ Sterilization of Glycerol and <br />
-
路Preparation of 25mg/mL kanamycin</td>
+
Preparation of 25mg/mL kanamycin</td>
-
<td>鈥ransformation of the parts mentioned on Jul.9th for the
+
<td>▪ransformation of the parts mentioned on Jul.9th for the
second time</td>
second time</td>
-
<td>鈥bservation the sections</td>
+
<td>▪bservation the sections</td>
</table>
</table>
</td>
</td>
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cellpadding="1">
cellpadding="1">
-
<td width="128">鈥?Pick two colonies of each parts and cultivate
+
<td width="128">▪ Pick two colonies of each parts and cultivate
them in LB medium</td>
them in LB medium</td>
-
<td width="203">鈥ransformation of three parts(20J,20H.22B from
+
<td width="203">▪ransformation of three parts(20J,20H.22B from
Kit plates of 2011 Distribution ) which are related to the
Kit plates of 2011 Distribution ) which are related to the
degradation of Cellulose</td>
degradation of Cellulose</td>
-
<td width="91">鈥⒙稭ini prep to isolate 10I,12I and 22M</td>
+
<td width="91">▪Min prep to isolate 10I,12I and 22M</td>
-
<td width="130">鈥onservation of 10I,12I,22M and 11P</td>
+
<td width="130">▪onservation of 10I,12I,22M and 11P</td>
</table>
</table>
</td>
</td>
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<table width="618" border="0" cellspacing="0" cellpadding="1">
<table width="618" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="104">鈥?Place the culture plate of 20J,20H and 22B in
+
<td width="104">▪ Place the culture plate of 20J,20H and 22B in
the fridge.</td>
the fridge.</td>
-
<td width="102">鈥in prep to isolate 13K 鈥onservation of 13K</td>
+
<td width="102">▪in prep to isolate 13K ▪onservation of 13K</td>
-
<td width="163">鈥estriction digest of the
+
<td width="163">▪estriction digest of the
parts(12I,10I,22M,13K) with EcoRI and PstI</td>
parts(12I,10I,22M,13K) with EcoRI and PstI</td>
-
<td width="111">鈥el electrophoresis to analyse restriction
+
<td width="111">▪el electrophoresis to analyse restriction
fragments</td>
fragments</td>
-
<td width="128">鈥est the Tm of Primers CP1&amp;CS,NP&amp;NS
+
<td width="128">▪est the Tm of Primers CP1&amp;CS,NP&amp;NS
-
with 13K. The result of Gel electrophoresis shows that 60.2鈩?is the
+
with 13K. The result of Gel electrophoresis shows that 60.2℃ is the
-
Tm of NS and NP, and 57.4鈩?is the Tm of CS and CP1.</td>
+
Tm of NS and NP, and 57.4℃ is the Tm of CS and CP1.</td>
</tr>
</tr>
</table>
</table>
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<table width="618" border="0" cellspacing="0" cellpadding="1">
<table width="618" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td>鈥?Test the Tm of the YFP,RFP,tetR,Vgb and nirB primers.
+
<td>▪ Test the Tm of the YFP,RFP,tetR,Vgb and nirB primers.
The result of Gel electrophoresis shows that the Tm for the primers
The result of Gel electrophoresis shows that the Tm for the primers
-
of Vgb is 54鈩?and the Tm for the primers of nirB is 55鈩?The RCR of
+
of Vgb is 54℃ and the Tm for the primers of nirB is 55℃ The RCR of
YFP,RFP and tetR failed.</td>
YFP,RFP and tetR failed.</td>
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<table width="600" border="0" cellspacing="0" cellpadding="1">
<table width="600" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="155">鈥?PCR(Phusion)<br />
+
<td width="155">▪ PCR(Phusion)<br />
-
鈥?Digest 10I, 22M, 13K 鈥?Used wrong cutter, digestion again.</td>
+
▪ Digest 10I, 22M, 13K ▪ Used wrong cutter, digestion again.</td>
-
<td>鈥?Run the results of PCR and the first digestion. The
+
<td>▪ Run the results of PCR and the first digestion. The
annealing temperature of YFP needed change. The digestion results
annealing temperature of YFP needed change. The digestion results
confirmed</td>
confirmed</td>
-
<td>鈥?Run the digestion results of second time. The bands are
+
<td>▪ Run the digestion results of second time. The bands are
confirmed.</td>
confirmed.</td>
</tr>
</tr>
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<table width="620" border="0" cellspacing="0" cellpadding="1">
<table width="620" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="127">鈥?Cut the linearized pSB1k3 with E+P</td>
+
<td width="127">▪ Cut the linearized pSB1k3 with E+P</td>
-
<td width="123">鈥?Purify the digestion results of 22M, 13K, 10I</td>
+
<td width="123">▪ Purify the digestion results of 22M, 13K, 10I</td>
-
<td width="133">鈥?Confirm digestion of pSB1k3 by
+
<td width="133">▪ Confirm digestion of pSB1k3 by
electrophoresis, then purification</td>
electrophoresis, then purification</td>
-
<td width="113">鈥?Test Tm of YFP<br />
+
<td width="113">▪ Test Tm of YFP<br />
-
鈥?ligation: 22M+10I, 13K+10I</td>
+
▪ ligation: 22M+10I, 13K+10I</td>
-
<td width="114">鈥?Tm of YFP is 54 degree 鈥?transform the
+
<td width="114">▪ Tm of YFP is 54 degree ▪ transform the
ligation results.</td>
ligation results.</td>
</tr>
</tr>
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<table width="620" border="0" cellspacing="0" cellpadding="1">
<table width="620" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td>鈥?PCR 22M<br />
+
<td>▪ PCR 22M<br />
-
鈥?purify Vgb, RFP, RFP-tetR, YFP-tetR. Gibson PCR.</td>
+
▪ purify Vgb, RFP, RFP-tetR, YFP-tetR. Gibson PCR.</td>
-
<td>鈥?ligation the purified the fragments in yesterday.</td>
+
<td>▪ ligation the purified the fragments in yesterday.</td>
-
<td>鈥?22M PCR<br />
+
<td>▪ 22M PCR<br />
-
鈥?Transform the ligation results.</td>
+
▪ Transform the ligation results.</td>
</tr>
</tr>
</table>
</table>
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cellpadding="1">
cellpadding="1">
-
<td width="128">鈥?Genome extraction of E.coli</td>
+
<td width="128">▪ Genome extraction of E.coli</td>
-
<td width="196">鈥?PCR of NirB<br />
+
<td width="196">▪ PCR of NirB<br />
-
鈥?cut 10I with X+P, cut 22M, 13K with E+S, cut pSB1k3 with E+P</td>
+
▪ cut 10I with X+P, cut 22M, 13K with E+S, cut pSB1k3 with E+P</td>
-
<td>鈥?Ligate: 10I+13K, 10I+22M <br />
+
<td>▪ Ligate: 10I+13K, 10I+22M <br />
-
鈥?Repeat NirB PCR</td>
+
▪ Repeat NirB PCR</td>
<td>
<td>
-
<p align="left">鈥?Culture 11P<br />
+
<p align="left">▪ Culture 11P<br />
-
鈥?Miniprep 10I, 22M, 13K</p>
+
▪ Miniprep 10I, 22M, 13K</p>
</td>
</td>
</table>
</table>
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cellpadding="1">
cellpadding="1">
-
<td width="140">鈥?mini-prep: 10I+13K, 10I+22M</td>
+
<td width="140">▪ mini-prep: 10I+13K, 10I+22M</td>
-
<td width="213">鈥nsert 10I+13K, 10I+22M into pSB1k3</td>
+
<td width="213">▪nsert 10I+13K, 10I+22M into pSB1k3</td>
-
<td width="110">鈥ransform: 5E, 3C, 7C, 1K, 1I from the
+
<td width="110">▪ransform: 5E, 3C, 7C, 1K, 1I from the
distribution plate</td>
distribution plate</td>
-
<td width="144">鈥ransform 10I+22M, 10I+13K</td>
+
<td width="144">▪ransform 10I+22M, 10I+13K</td>
</table>
</table>
</td>
</td>
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<table width="610" border="0" cellspacing="0" cellpadding="1">
<table width="610" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="170">鈥olonies of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K,
+
<td width="170">▪olonies of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K,
1I confirmed</td>
1I confirmed</td>
-
<td width="102">鈥?Gibson PCR</td>
+
<td width="102">▪ Gibson PCR</td>
-
<td width="250">鈥olony PCR 22M+10I, 13K+10I, 5E, 3C, 7C, 1K,
+
<td width="250">▪olony PCR 22M+10I, 13K+10I, 5E, 3C, 7C, 1K,
1I</td>
1I</td>
-
<td width="80">鈥CR NirB</td>
+
<td width="80">▪CR NirB</td>
</tr>
</tr>
</table>
</table>
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<table width="618" border="0" cellspacing="0" cellpadding="1">
<table width="618" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td>鈥CR verification of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I
+
<td>▪CR verification of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I
</td>
</td>
-
<td>鈥?Gibson PCR 鈥?Run the results of PCR verification. Bands
+
<td>▪ Gibson PCR ▪ Run the results of PCR verification. Bands
confirmed.</td>
confirmed.</td>
-
<td>鈥?Culture the 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I 鈥?
+
<td>▪ Culture the 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I ▪
Purification of Gibson PCR results</td>
Purification of Gibson PCR results</td>
</tr>
</tr>
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<table width="618" border="0" cellspacing="0" cellpadding="1">
<table width="618" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="117">鈥?PCR amplification of Gibson assembly results
+
<td width="117">▪ PCR amplification of Gibson assembly results
</td>
</td>
-
<td width="144">鈥?Mini prep: 22M+10I, 22.Presever in -20<br />
+
<td width="144">▪ Mini prep: 22M+10I, 22.Presever in -20<br />
-
鈥?Medi prep: 1K,1I,3C,5E,7C<br />
+
▪ Medi prep: 1K,1I,3C,5E,7C<br />
-
鈥?Gibson Assembly fail.</td>
+
▪ Gibson Assembly fail.</td>
-
<td width="164">鈥?PCR NirB by Phusion<br />
+
<td width="164">▪ PCR NirB by Phusion<br />
-
鈥epeat PCR by changing Pnibr to Gnirbr<br />
+
▪epeat PCR by changing Pnibr to Gnirbr<br />
-
鈥ut the mini and medi prep results with E</td>
+
▪ut the mini and medi prep results with E</td>
-
<td width="185">鈥un the results of PCR and digestion. Fail in
+
<td width="185">▪un the results of PCR and digestion. Fail in
PCR of NirB, succeed in 10I+3K and 10I+22M ligation.</td>
PCR of NirB, succeed in 10I+3K and 10I+22M ligation.</td>
</tr>
</tr>
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<table width="613" border="0" cellspacing="0" cellpadding="1">
<table width="613" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="237">鈥ouble digestion of 13K+10I, 22M+10I, pSB1c3
+
<td width="237">▪ouble digestion of 13K+10I, 22M+10I, pSB1c3
</td>
</td>
-
<td width="169">鈥?Fail in purification of Medi prep</td>
+
<td width="169">▪ Fail in purification of Medi prep</td>
-
<td width="201">鈥?PCR NirB<br />
+
<td width="201">▪ PCR NirB<br />
-
鈥?Ligate NirB+13K+10I, Vgb+22M+10I</td>
+
▪ Ligate NirB+13K+10I, Vgb+22M+10I</td>
</tr>
</tr>
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<table width="608" border="0" cellspacing="0" cellpadding="1">
<table width="608" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="244">鈥?Gibson assembly: NirB+RFP+tetR<br />
+
<td width="244">▪ Gibson assembly: NirB+RFP+tetR<br />
-
鈥?Cut results of Gibson assembly and pSB1c3.</td>
+
▪ Cut results of Gibson assembly and pSB1c3.</td>
-
<td width="164">鈥?Purify the ligation results in yesterday</td>
+
<td width="164">▪ Purify the ligation results in yesterday</td>
-
<td width="194">鈥?ligate with backbone 鈥?Culture 1I, 1K, 3C,
+
<td width="194">▪ ligate with backbone ▪ Culture 1I, 1K, 3C,
5E, 7C, 11P</td>
5E, 7C, 11P</td>
</tr>
</tr>
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cellpadding="1">
cellpadding="1">
-
<td width="128">鈥?Transform Pnirb+13k+10I+pSBK3-2</td>
+
<td width="128">▪ Transform Pnirb+13k+10I+pSBK3-2</td>
-
<td>鈥?Place the culture in 4 degree</td>
+
<td>▪ Place the culture in 4 degree</td>
-
<td>鈥?Medi-and mini-prep of five cultre</td>
+
<td>▪ Medi-and mini-prep of five cultre</td>
-
<td>鈥ibson Assembly confirmation by gel</td>
+
<td>▪ibson Assembly confirmation by gel</td>
</table>
</table>
</td>
</td>
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cellpadding="1">
cellpadding="1">
-
<td width="134">鈥?Cut Pnirb, Pvgb,(E+S)<br />
+
<td width="134">▪ Cut Pnirb, Pvgb,(E+S)<br />
-
鈥?Cut 10I+13K, 10I+22M.(X+P)</td>
+
▪ Cut 10I+13K, 10I+22M.(X+P)</td>
-
<td width="167">鈥?Ligation: vgb+10I+22M, nirB+10I+13K,</td>
+
<td width="167">▪ Ligation: vgb+10I+22M, nirB+10I+13K,</td>
-
<td width="172">鈥?Ligation: pSBK13+vgb+10I+22M,
+
<td width="172">▪ Ligation: pSBK13+vgb+10I+22M,
pSBK13+nirB+10I+13K</td>
pSBK13+nirB+10I+13K</td>
-
<td width="129">鈥?Transform the ligation results.</td>
+
<td width="129">▪ Transform the ligation results.</td>
</table>
</table>
</td>
</td>
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<table width="608" border="0" cellspacing="0" cellpadding="1">
<table width="608" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="170">鈥?No colony on the plate<br />
+
<td width="170">▪ No colony on the plate<br />
-
鈥?Gibson assembly</td>
+
▪ Gibson assembly</td>
-
<td width="184">鈥?Run the results of Gibson assembly</td>
+
<td width="184">▪ Run the results of Gibson assembly</td>
-
<td width="248">鈥?Excise the bands, the purify the DNA<br />
+
<td width="248">▪ Excise the bands, the purify the DNA<br />
-
鈥?Gibson Assenmly</td>
+
▪ Gibson Assenmly</td>
</tr>
</tr>
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<table width="609" border="0" cellspacing="0" cellpadding="1">
<table width="609" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="607">鈥?011 China Meet-up @ Hefei</td>
+
<td width="607">▪ 011 China Meet-up @ Hefei</td>
</tr>
</tr>
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<table width="607" border="0" cellspacing="0" cellpadding="1">
<table width="607" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td>鈥?011 China Meet-up @ Hefei</td>
+
<td>▪ 011 China Meet-up @ Hefei</td>
</tr>
</tr>
</table>
</table>
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<table width="600" border="0" cellspacing="0" cellpadding="1">
<table width="600" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="368">鈥?Culture 22M+10I, 13K+10I</td>
+
<td width="368">▪ Culture 22M+10I, 13K+10I</td>
-
<td width="228">鈥?Cut a0H, 20J, 22B</td>
+
<td width="228">▪ Cut a0H, 20J, 22B</td>
</tr>
</tr>
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<tr>
<tr>
<td>Plasmid</td>
<td>Plasmid</td>
-
<td>1渭L (>100ng)</td>
+
<td>1μL (>100ng)</td>
</tr>
</tr>
<tr>
<tr>
<td>EcoRI</td>
<td>EcoRI</td>
-
<td>1渭L</td>
+
<td>1μL</td>
</tr>
</tr>
<tr>
<tr>
<td>PstI</td>
<td>PstI</td>
-
<td>1渭L</td>
+
<td>1μL</td>
</tr>
</tr>
<tr>
<tr>
-
<td>10脳Buffer Tango</td>
+
<td>10×Buffer Tango</td>
-
<td>2渭L</td>
+
<td>2μL</td>
</tr>
</tr>
<tr>
<tr>
<td>ddH2O</td>
<td>ddH2O</td>
-
<td>15渭L</td>
+
<td>15μL</td>
</tr>
</tr>
</table>
</table>
Line 655: Line 655:
width="200" height="200" /></p>
width="200" height="200" /></p>
<p><strong>Temperature grad</strong><br />
<p><strong>Temperature grad</strong><br />
-
56鈩?57.4鈩?60.2鈩?62.9鈩?64.3鈩?65.8鈩?/p>
+
56℃ 57.4℃ 60.2℃ 62.9℃ 64.3℃ 65.8℃ /p>
<div id="framecontent">
<div id="framecontent">
<p>&nbsp;</p>
<p>&nbsp;</p>
Line 665: Line 665:
cellpadding="1">
cellpadding="1">
<tr>
<tr>
-
<td>10脳Buffer</td>
+
<td>10×Buffer</td>
-
<td>2渭L</td>
+
<td>2μL</td>
</tr>
</tr>
<tr>
<tr>
<td>dNTPs</td>
<td>dNTPs</td>
-
<td>0.5渭L</td>
+
<td>0.5μL</td>
</tr>
</tr>
<tr>
<tr>
<td>primers CP1 (NP)</td>
<td>primers CP1 (NP)</td>
-
<td>1渭L</td>
+
<td>1μL</td>
</tr>
</tr>
<tr>
<tr>
<td>primers CS (NS)</td>
<td>primers CS (NS)</td>
-
<td>1渭L</td>
+
<td>1μL</td>
</tr>
</tr>
<tr>
<tr>
<td>Template</td>
<td>Template</td>
-
<td>1渭L</td>
+
<td>1μL</td>
</tr>
</tr>
<tr>
<tr>
<td>rTaq</td>
<td>rTaq</td>
-
<td>0.2渭L</td>
+
<td>0.2μL</td>
</tr>
</tr>
<tr>
<tr>
<td>H2O</td>
<td>H2O</td>
-
<td>14.5渭L</td>
+
<td>14.5μL</td>
</tr>
</tr>
<tr>
<tr>
<td>Total</td>
<td>Total</td>
-
<td>20渭L</td>
+
<td>20μL</td>
</tr>
</tr>
</table>
</table>
Line 738: Line 738:
<div class="block" id="nsheet">
<div class="block" id="nsheet">
<h1>28th July</h1>
<h1>28th July</h1>
-
<p>13.30: DH5蓱 11p 5mlX4<br />
+
<p>13.30: DH5α 11p 5mlX4<br />
-
23.00: silicone tube set at 37鈩?/p>
+
23.00: silicone tube set at 37℃ /p>
</div>
</div>
<div class="block" id="nsheet">
<div class="block" id="nsheet">
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<div class="block" id="nsheet">
<h1>31st July</h1>
<h1>31st July</h1>
-
<p>13.00: -80鈩?storing silicone tube. A thick white and loosely bond
+
<p>13.00: -80℃ storing silicone tube. A thick white and loosely bond
substance is seen on the inner wall of the 5mm silicone tube, and on the
substance is seen on the inner wall of the 5mm silicone tube, and on the
inner wall of the 1mm silicone tube a flatter and smoother white
inner wall of the 1mm silicone tube a flatter and smoother white
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Revision as of 08:59, 17 October 2011

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> Notebook - July

Lab Notes

July August
 September

Protocol

Protocol

Brainstorm

Brainstorm

Lab Notes - July

Week1

Day Note
Jul.4th Monday
▪Aerobic cultivation of DH5α ▪ Preparation of apparatus for the formation of biofilm
Jul 5th Tuesday  
Jul.6th Wednesday
▪eceiving primers ordered previously
Jul.7th Thursday
▪ Preparation of the aliquot of the primers ▪ Something wrong with a shaking incubator
Jul.8th Friday
▪ Preparation of culture plates for the transformations
Jul.9th Saturday
▪ Preparation of culture plates for the transformations ▪ protocols of transformation
Jul.10th Sunday
▪ Several colonies were picked up and cultivated in 5mL LB medium. ▪ryosectioning of biofilm

Week2

Day Note
Jul.11th Monday
▪ Set up new LB culture plates with ampicillin and kanamycin

▪ Sterilization of Glycerol and
Preparation of 25mg/mL kanamycin		 ▪ransformation of the parts mentioned on Jul.9th for the second time ▪bservation the sections
Jul 12th Tuesday
▪ Pick two colonies of each parts and cultivate them in LB medium ▪ransformation of three parts(20J,20H.22B from Kit plates of 2011 Distribution ) which are related to the degradation of Cellulose ▪Min prep to isolate 10I,12I and 22M ▪onservation of 10I,12I,22M and 11P
Jul.13th Wednesday
▪ Place the culture plate of 20J,20H and 22B in the fridge. ▪in prep to isolate 13K ▪onservation of 13K ▪estriction digest of the parts(12I,10I,22M,13K) with EcoRI and PstI ▪el electrophoresis to analyse restriction fragments ▪est the Tm of Primers CP1&CS,NP&NS with 13K. The result of Gel electrophoresis shows that 60.2℃ is the Tm of NS and NP, and 57.4℃ is the Tm of CS and CP1.
Jul.14th Thursday
▪ Test the Tm of the YFP,RFP,tetR,Vgb and nirB primers. The result of Gel electrophoresis shows that the Tm for the primers of Vgb is 54℃ and the Tm for the primers of nirB is 55℃ The RCR of YFP,RFP and tetR failed.
Jul.15th Friday
▪ PCR(Phusion)
▪ Digest 10I, 22M, 13K ▪ Used wrong cutter, digestion again.		 ▪ Run the results of PCR and the first digestion. The annealing temperature of YFP needed change. The digestion results confirmed ▪ Run the digestion results of second time. The bands are confirmed.
Jul.16th Saturday
▪ Cut the linearized pSB1k3 with E+P ▪ Purify the digestion results of 22M, 13K, 10I ▪ Confirm digestion of pSB1k3 by electrophoresis, then purification ▪ Test Tm of YFP
▪ ligation: 22M+10I, 13K+10I		 ▪ Tm of YFP is 54 degree ▪ transform the ligation results.
Jul.17th Sunday
▪ PCR 22M
▪ purify Vgb, RFP, RFP-tetR, YFP-tetR. Gibson PCR.		 ▪ ligation the purified the fragments in yesterday. ▪ 22M PCR
▪ Transform the ligation results.

 

Week3

Day Note
Jul.18th Monday
▪ Genome extraction of E.coli ▪ PCR of NirB
▪ cut 10I with X+P, cut 22M, 13K with E+S, cut pSB1k3 with E+P		 ▪ Ligate: 10I+13K, 10I+22M
▪ Repeat NirB PCR

▪ Culture 11P
▪ Miniprep 10I, 22M, 13K
Jul 19th Tuesday
▪ mini-prep: 10I+13K, 10I+22M ▪nsert 10I+13K, 10I+22M into pSB1k3 ▪ransform: 5E, 3C, 7C, 1K, 1I from the distribution plate ▪ransform 10I+22M, 10I+13K
Jul.20th Wednesday
▪olonies of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I confirmed ▪ Gibson PCR ▪olony PCR 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I ▪CR NirB
Jul.21th Thursday
▪CR verification of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I ▪ Gibson PCR ▪ Run the results of PCR verification. Bands confirmed. ▪ Culture the 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I ▪ Purification of Gibson PCR results
Jul.22th Friday
▪ PCR amplification of Gibson assembly results ▪ Mini prep: 22M+10I, 22.Presever in -20
▪ Medi prep: 1K,1I,3C,5E,7C
▪ Gibson Assembly fail.		 ▪ PCR NirB by Phusion
▪epeat PCR by changing Pnibr to Gnirbr
▪ut the mini and medi prep results with E		 ▪un the results of PCR and digestion. Fail in PCR of NirB, succeed in 10I+3K and 10I+22M ligation.
Jul.23th Saturday
▪ouble digestion of 13K+10I, 22M+10I, pSB1c3 ▪ Fail in purification of Medi prep ▪ PCR NirB
▪ Ligate NirB+13K+10I, Vgb+22M+10I
Jul.24th Sunday
▪ Gibson assembly: NirB+RFP+tetR
▪ Cut results of Gibson assembly and pSB1c3.		 ▪ Purify the ligation results in yesterday ▪ ligate with backbone ▪ Culture 1I, 1K, 3C, 5E, 7C, 11P

 

Week4

Day Note
Jul.25th Monday
▪ Transform Pnirb+13k+10I+pSBK3-2 ▪ Place the culture in 4 degree ▪ Medi-and mini-prep of five cultre ▪ibson Assembly confirmation by gel
Jul. 26th Tuesday
▪ Cut Pnirb, Pvgb,(E+S)
▪ Cut 10I+13K, 10I+22M.(X+P)		 ▪ Ligation: vgb+10I+22M, nirB+10I+13K, ▪ Ligation: pSBK13+vgb+10I+22M, pSBK13+nirB+10I+13K ▪ Transform the ligation results.
Jul.27th Wednesday
▪ No colony on the plate
▪ Gibson assembly		 ▪ Run the results of Gibson assembly ▪ Excise the bands, the purify the DNA
▪ Gibson Assenmly
Jul.28th Thursday
▪ 011 China Meet-up @ Hefei
Jul.29th Friday
▪ 011 China Meet-up @ Hefei
Jul.30th Saturday
Jul.31th Sunday
▪ Culture 22M+10I, 13K+10I ▪ Cut a0H, 20J, 22B


Data/Protocol

Jul.13th Wednesday

Systems of restriction digestion with EcoRI and PstI

Plasmid 1μL (>100ng)
EcoRI 1μL
PstI 1μL
10×Buffer Tango 2μL
ddH2O 15μL

Temperature grad
56℃ 57.4℃ 60.2℃ 62.9℃ 64.3℃ 65.8℃ /p>

 

PCR system (test the Tm of the primers CP1&CS, NP&NS)

10×Buffer 2μL
dNTPs 0.5μL
primers CP1 (NP) 1μL
primers CS (NS) 1μL
Template 1μL
rTaq 0.2μL
H2O 14.5μL
Total 20μL

Within each compartment are components: include different types of biomass ,substrates , products. biomass is often divided into active microbial species, inert cells, and extracellular polymeric substances(EPS).

The components can undergo transformation, transport, and transfer processes. For example, substrate is consumed, and this leads to the synthesis of new active biomass.

All process affecting each component in each compartment are mathematically linked together into a mass balance equation that contains rate terms and parameters for each process.

Model Selection:Many kinds of Mathematics models have been founded to describe a system of biofilm. Models of different dimensions (1d, 2d, 3d) focus on different properties of a biofilm. Since we care most about the oxygen concentration gradients perpendicular to the substratum, numerical 1-dimensional dynamic model(N1) would be a proper choice for us.

 

Early July

Pre-experiments with biofilm formation with circular and non circular silicone tube, 24 well plate on different support including glass, paper, plastic film, rubber. The final material are used based on the easiness biofilm form on them and on the easiness to observe under microscope.

 

28th July

13.30: DH5α 11p 5mlX4
23.00: silicone tube set at 37℃ /p>

29th July

13.00: LB culture 50ml with circular culture medium. LB culture 50ml with noncircular culture medium.

31st July

13.00: -80℃ storing silicone tube. A thick white and loosely bond substance is seen on the inner wall of the 5mm silicone tube, and on the inner wall of the 1mm silicone tube a flatter and smoother white substance. Especially obvious where the tube turns. Possibly because the speed of culture flow is slower.

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