Team:ZJU-China/Notebook

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<<div  class="pagetitle" id="nbook">
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      <table style="background-color:transparent;" width="750" border="0" cellspacing="0" cellpadding="1">
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     <td><div class="pagetitle" id="stra1"><a> <h3>Biobrick</h3>  
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   <tr>
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          <p>brief introduction</p></a></div></td>  
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     <td ><h3>Modeling|biobrick</h3></td>
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     <td><div class="pagetitle" id="stra2"><a href="https://2011.igem.org/Team:ZJU-China/Notebook/Biofilm"> <h3>Biofilm Formation</h3>  
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    <td > <div class="roundcorner" >
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          <p>brief introduction</p></a></div></td>  
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     <p><a name="jumptop" id="p_intro">&nbsp;Introduction&nbsp;</a>|<a id="p_model">&nbsp;Model&nbsp;</a>|<a id="p_simul">&nbsp;Simulation&nbsp;</a></p>
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                <ul class="kwicks">  
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<p style="margin-left:10px;">This model is used for simulating the behavior of three genetic circuits we designed</p>
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                    <li id="kwick1"><a>week1</a><p>In this week we........</p></li>  
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                    <li id="kwick2"><a>week2</a></li>  
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                    <li id="kwick3"><a>week3</a></li>  
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                    <li id="kwick4"><a>week4</a></li>  
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                    <li id="kwick5"><a>week5</a></li>  
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                    <li id="kwick6"><a>week6</a></li>  
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    <div id="leftul"><div class="leftul">  
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                    <li id="kwick7"><a>week7</a></li>  
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                    <li id="kwick8"><a>week8</a></li>  
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                    <li id="kwick9"><a>week9</a></li>  
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<h4>Overview</h4>
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                    <li id="kwick10"><a>week10</a></li>  
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                    <li id="kwick11"><a>week11</a></li>  
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<div class="pane" >  
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                    <li id="kwick12"><a>week12</a></li>  
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                <a  href="https://2011.igem.org/Team:ZJU-China/Project">Summary</a>
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                    <li id="afkwick12"><a style="font-size:18pt;margin:1px;padding:1px;">after week12</a></li>  
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<a  href="https://2011.igem.org/Team:ZJU-China/Project/Stratifiedsystem">Background<br/></a>
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                 </ul>  
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<a  href="https://2011.igem.org/Team:ZJU-China/Project/Result">Results<br/></a>
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            </div>  
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        <a  href="https://2011.igem.org/Team:ZJU-China/Project/Future">Future Application</a>
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<h4 class="current">Modeling</h4>  
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<a href="https://2011.igem.org/Team:ZJU-China/Modeling">|Biofilm formation<br/></a>
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<a  href="https://2011.igem.org/Team:ZJU-China/Modeling/Biobrick">|Biobrick<br/></a>
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        <a  href="#">|Results<br/></a>
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<h4>Results</h4>  
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<div class="pane" >  
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<a href="https://2011.igem.org/Team:ZJU-China/Datapage">Data page</a>
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                <a  href="https://2011.igem.org/Team:ZJU-China/Parts">Parts</a>
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<div class="bigblock"><div class="inner" id="week1">  
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Revision as of 16:45, 2 October 2011

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Modeling|biobrick

 Introduction | Model | Simulation 

This model is used for simulating the behavior of three genetic circuits we designed

Week1

DayNote
Jul.4th Monday
• Aerobic cultivation of DH5α • Preparation of apparatus for the formation of biofilm
Jul 5th Tuesday  
Jul.6th Wednesday
•Receiving 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. •Cryosectioning of biofilm

 

Data&protocol

Monday

blanknote

There is no data in first week

 

Week2

DayNote
Jul.11th Monday
• Set up new LB culture plates with ampicillin and kanamycin

 • ·Sterilization of Glycerol and
·Preparation of 25mg/mL kanamycin		 •Transformation of the parts mentioned on Jul.9th for the second time •Observation the sections
Jul 12th Tuesday
• Pick two colonies of each parts and cultivate them in LB medium •Transformation of three parts(20J,20H.22B from Kit plates of 2011 Distribution ) which are related to the degradation of Cellulose •·Mini prep to isolate 10I,12I and 22M •Conservation of 10I,12I,22M and 11P
Jul.13th Wednesday
• Place the culture plate of 20J,20H and 22B in the fridge. •Min prep to isolate 13K •Conservation of 13K •Restriction digest of the parts(12I,10I,22M,13K) with EcoRI and PstI •Gel electrophoresis to analyse restriction fragments •Test 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.

 

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℃

 

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

Jul.14th Thursday

PCR systems(test the Tm of the primers for YFP,RFP,tetR,Vgb and nirB )

10×Buffer 2μL
dNTPs 0.5μL
primers F 0.5μL
primers R 0.5μL
Template 1μL
Taq 0.2μL
H2O 15.5μL
Total 20μL

 

Temperature grad and the results:

vgb&nirB pcr test:

 

 

Week3

DayNote
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 •insert 10I+13K, 10I+22M into pSB1k3 •transform: 5E, 3C, 7C, 1K, 1I from the distribution plate •transform 10I+22M, 10I+13K
Jul.20th Wednesday
•Colonies of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I confirmed • Gibson PCR •Colony PCR 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I •PCR NirB
Jul.21th Thursday
•PCR 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
•Repeat PCR by changing Pnibr to Gnirbr
•Cut the mini and medi prep results with E 		•Run the results of PCR and digestion. Fail in PCR of NirB, succeed in 10I+3K and 10I+22M ligation.
Jul.23th Saturday
•Double 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

 

Data&protocol

Monday

WEEK3

Tuesday

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Week4

DayNote
Jul.25th Monday
• Transform Pnirb+13k+10I+pSBK3-2 • Place the culture in 4 degree • Medi-and mini-prep of five cultre •Gibson 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
•2011 China Meet-up @ Hefei
Jul.29th Friday
•2011 China Meet-up @ Hefei
Jul.30th Saturday
Jul.31th Sunday
• Culture 22M+10I, 13K+10I • Cut a0H, 20J, 22B

 

Data&protocol

Monday

WEEK3

Tuesday

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Week5

DayNote
Aug.1st Monday
•Fusion PCR Vgb+YFP+tetR, NirB+RFP+tetR, •PCR: NirB, Vgb
Aug.2nd Tuesday
•Cut: 13K+10I, 22M+10I • Purify: 13K+10I, 22M+10I
• Ligation: Pvgb+22M+10I, Pnirb+13K+10I 		• PCR backbones
• Electrophresis 		• Gel excision and purification
Aug.3rd Wednesday
• Make Phusion Buffer, 5×isothermel buffer • colony PCR: 20H, 20J, 22B
Aug.4th Thursday
• Cut: 10I+22M, 10I+13; and purification • Ligation: Pvgb+22M+10I, Pnirb+13K+10I,
• colony PCR: 13K+10I 		• Culture: 20H, 20J, 22B, 1K, 1I,3C, 5E, 7C • Transform: 1G, 3A, 5A, 7A from the distribution plate
Aug.5th Friday
• Check the plates. Contamination, or no positive colonies • Miniprep: 5 backbones, 22B-1, 22B-3 • PCR: nirB from 13K+10I+nirB to firm the ligation. One positive result. • Culture the positive colony.
Aug.6th Saturday
Aug.7th Sunday
• Cut: 22M,13K with E & S • Culture the red colonies from plate of pSB1C3 • PCR: 5 backbones
• Cut the PCR products with P+E and run the gel to confirm.

Data&protocol

Monday

WEEK3

Tuesday

bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

 

Week6

DayNote
Aut.8th Monday
• The gel results of 5 backbones are right. • Miniprep: pSB1C3, vgb+22M+10I • Culture vgb+22M+1oI in hypoxia
•PCR pSB1C3
Aug.9th Tuesday
• Run the PCR product • PCR pSB1C3 again • Run the product, results are good.
Aug.10th Wednesday
• Culture: 20H, 20J
• Transform 13K from plate 3 		• Miniprep: 20H-1, 20J-1 • Cut: the 20H-1, 20J-1, 22B-1, 22B-3 with E+P
Aug.11th Thursday
• Run the digestion results. Bands are confirmed right. • Colony PCR vgb+22M+10I • Cut the plasmid of vgb+22M+10I
Aug.12th Friday
• Make new stock of competent cells • Ligation: 13K+10I
• Cut the PCR results and 22M with E+P 		• Sequence: nirB, vgbL, 12I, 20H, 20J, 22B • Transform 13K+10I+pSB1K3
• Cut 13K to validate. Results are right.
Aug.13th Saturday
• Colony PCR: 13K+10I+pSB1K3 • Culture: vgb+22M+10I in 5ml(shaking), 5ml(water bath), 10ml(water bath), 15(water bath). • Check the YFP expression of 5ml(shaking) and 15ml(water bath). No yellow fluorescent cells,
Aug.14th Sunday
• Transform: the Gibson assembly results. • Miniprep: 13K+10I
• Cut: 13K+10I 		• Purification: the digestion results.
• Ligation: nirB+13K+10I

Data&protocol

Monday

WEEK3

Tuesday

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Week7

DayNote
Aug.15th Monday
• Plate: nirB+13K+10I • Colony PCR: vgb+YFP+tetR +terminater • Gibson PCR
Aug.16th Tuesday
• Colony PCR: vgb+22M+10I, nirB+13K+10I • No bands of 13K; 22M confirmed
Aut.17th Wednesday
• Miniprep: 22M
• Cut: 22M 		• Check CFP expression. No fluorescence.
Aug.18th Thursday
• Transform: 13K, 10I, 22M, 12I, 18P
Aug.19th Friday
• Miniprep: 13K, 10I, 22M, 12I, 18P • Cut: 13K, 10I, 22M, 12I, 18P • Run the gel
Aug.20th Saturday
• Ligate 13K+10I, 22M+10I
Aug.21st Sunday
• Transform: the Gibson assembly results. • Miniprep: 13K+10I
• Cut: 13K+10I 		• Purification: the digestion results.
• Ligation: nirB+13K+10I

Data&protocol

Monday

WEEK7

Tuesday

bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

 

Week8

DayNote
Aug. 22nd Monday
• Transform 13K+10I, 22M+10I • Test new primers.
• PCR: 22M+10I, 13K+10I 		• Run the PCR products.
• Cut the PCR products.
Aug. 23rd Tuesday
• Test primers: CS/CP, VF/VR, pSB1_f/r • Cut: 11P, 1F, 22M
• Run the cut results.1F-1/2/3 are right. 		• Mix the 1F-1/2 purification products • Cut: 1F-1+1F-2(E+S), 22M-3(X+P), pSB1C3(E+P)
Aug. 24th Wednesday
Aug. 25th Thursday
Aug. 26th Friday
• Amplify: vgb, 1C3
Aug.27th Saturday
• Digest: Vgb, 1C3, fdfhF, 22M+10I, 13K+10I
Aug.28th Sunday
• Ligate: vgb+22M+10I, fdfhF+13K+10I • Transform the ligation results.

Data&protocol

Monday

WEEK8

Tuesday

bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

 

Week9

DayNote
Aug.29th Monday
• Meeting. Summarize all the experiment work so far. • Cut: 22M(X+P), 1C3(E+P)
Aug.30th Tuesday
Aug.31st Wednesday
• Cut vgb+YFP+tetR, fdhF+RFP+tetR.
• Run the digestion results		 • Culture the positive results
Sept.1st Thursday
• Miniprep: Y#4, R#2/3/4/5
• Cut Y#4, R#2/3/4/5 		•Run the gel.
• Send R#3,Y#4 sample to sequencing 		• Hypoxia culture: RFP3, RFP4
• Preserve Y#4, R#2/3/4/5
Sept.2nd Friday
• Phusion PCR • Hypoxia Culture: R, Y
• Run the PCR results. Bands are confirmed right. 		• Purification: vgb, YFP, tetR.
Sept.3rd Saturday
• Culture: YFP, RFP, A25
• Culture in micro-oxygen: YFP, RFP, A25 		• Culture in slope medium: RFP, YFP, A25
• Hypoxia culture: RFP, YFP, A25 		• Check the fluorescence.
• Neither RFP in hypoxia or oxygen condition. Little YFP both in hypoxia and oxygen.
Sept.4th Sunday
• Miniprep: RFP3, YFP4 • Cut: RFP3, YFP4, !C3.

Data&protocol

Monday

WEEK9

Tuesday

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Week10

DayNote
Sept.5th Monday
• Run the gel: Yu’s PCR results, 1C3, RFP3, YFP4
• Purification: RFP3, YFP4 		• Ligation: RFP3+1C3, YFP4+1C3
• Hypoxia culture: RFP3, YFP4, A25. 		• Transform the ligation results.
• Confirmed RFP expression only in hypoxia condition.
Sept.6th Tuesday
• Check the RFP, YFP plates. • Colony PCR
Sept.7th Wednesday
• Culture YFP1/2, RFP3/4
Sept.8th Thurday
• Preserve the strain of RFP1, YFP1/2 in 1C3.
• Miniprep the remains. 		• Cut: YFP1/2, RFP1 • Run the digestion results.
• Culture RFP2
Sept.9th Friday
• Miniprep: RFP2, YFP2, RFP1 • Cut them and run the gel.
Sept.10th Saturday
• Cut YFP(1A3), RFP(1A3) with S+E • Purification: RFP(1A3), YFP(1A3) • Ligation: RFP+1C3, YFP+1C3 • Transform the ligation results.
Sept.11th Sunday
• Check the plates. • Culture lig RFY+YFP+IC3
• Culture 1K3 		• Colony PCR: RFP(1C3)， YFP(1C3)

Data&protocol

Monday

WEEK10

Tuesday

bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

 

Week11

DayNote
Sept.12th Monday
• Colony PCR: 1A3, 1K3, lig RFP+YFP+1C3. • Run the PCR results. • Miniprep: lig RFP+YFP+1C3
Sept.13th Tuesday
• Transform 2M, 2I, 6I, 23L. • Purification: 1K3
Sept.14th Wednesday
• Miniprep: 20H, 20J, 22B • Cut: 1K3 with E+P, lig3 with S+E, 12I(CFP) with P+X, • Gel excision and /or purification: 1K3, lig3, 12I(CFP) • Run the gel.
• Culture: 1I, 1K, 3L, 5E, 7C 		• Miniprep: RFP+YFP+1C3.
• Run the gel of 1K3, lig3, CFP again. 		• Gel excision and purification
• Ligation: Lig3+CFP+1K3
Sept.15th Thursday
• Preserve: 1I, 1K, 3C, 5E, 7C
• Transform the ligation results 		• Colony PCR: 2M, 2I，6I，23L
• Cut: 20H, 20J, 22B with X+P 		• Miniprep: 2M-1, 2I-1, 6I-1, 23L-1
• Cut: 2M, 2I with E+S
Sept.16th Friday
• Colony PCR: RFP+YFP+1K3 • Cut 13K with E+S, 10I with P+X, 1C3 with E+P • Run the gel: 13K, 10I, 1C3, 2I, 2M, 20J, 22H, 22B • Ligation: 13K+10I+1C3
• Culture: YFP(Amp), CFP(Amp)
Sept.17th Saturday
• Order primers for 20H, 20J
• Culture 20H-1, 20J-1, 22B-1 		• Plate the ligation results
• Culture RFP+YFP+CFP+1K3 		• Colony PCR: 3A, 1C3
• Preserve: 1K3, RFP+YFP+1C3.
Sept.18th Sunday
• Miniprep: 20J, 20H, RFP+YFP+CFP+1K3 1A3 • Run the gel of PCR results • Cut RFP+YFP+CFP+1K3 with E+P
• Colony PCR: 13K+10I+1C3 		• PCR: fdhF+rush+Vgb+tetR
• Preserve: 20H, 20J, 22B 		• Run the gel: 20H, 20J, 22B
• Cut the bands and purification. 		• Run the gel: 13K+10I+1C3, RFP+YFP+CFP+1K3
• Culture: 13K+10I+1C3
• Grad PCR: fdhF.

Data&protocol

Monday

WEEK11

Tuesday

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Week12

DayNote
Sept.19th Monday
• Miniprep: 13K+10I+1C3, 1C3
• Run the gel of digestion results. 		• Cut: 13K+10I+1C3, 1A3, fdhF • Run the gel: 20H, 20J, rush+vgb, RYC+1K3, fdhF, 13K+10I, 1A3 • Cut and purification: fdhF, 13K+10I, 1A3
• Culture: CFP, YFP 		• Purification: Rush PCR
• Cut: 22B with X, rush PCR results with S
Sept.20th Tuesday
• Run the 20H, 20J from PCR results.
• Transform: fdhF+13K+10I+1A3 		• Purification: 20B, Rush
• Ligation: 22B+Rush 		• Purification: ligation results
• Cut: the ligation results with X+S 		• PCR 20H
• Self-ligation: rush+22M
Sept.21st Wednesday
• Colony PCR: fdhF+13K+10I+1A3 on plates • Run the gel of PCR results • Culture the positive results.
Sept.22nd Thursday
• Miniprep: fdhF+13K+10I+1A3
• Preserve the above. 		• Cut and run the results: fdhF+13K+10I+1A3, 13K+10I+1C3 • Cut: RFP with E+S, YFP with X+P, 1K3 with E+P, 1C3 with E+P • Purification the digestion results.
Sept.23rd Friday
• Ligate: RFP+1A3/ YFP+1C3/1K3, RFP+1A3/1C3 • Transform the ligation results.
Sept. 24th Saturday
• Order primers for 20H, 20J
• Culture 20H-1, 20J-1, 22B-1 		• Plate the ligation results
• Culture RFP+YFP+CFP+1K3 		• Colony PCR: 3A, 1C3
• Preserve: 1K3, RFP+YFP+1C3.
Sept.25th Sunday
• Miniprep: 20J, 20H, RFP+YFP+CFP+1K3 1A3 • Run the gel of PCR results • Cut RFP+YFP+CFP+1K3 with E+P
• Colony PCR: 13K+10I+1C3 		• PCR: fdhF+rush+Vgb+tetR
• Preserve: 20H, 20J, 22B 		• Run the gel: 20H, 20J, 22B
• Cut the bands and purification. 		• Run the gel: 13K+10I+1C3, RFP+YFP+CFP+1K3
• Culture: 13K+10I+1C3
• Grad PCR: fdhF.

Data&protocol

Monday

WEEK12

Tuesday

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Data&protocol

Monday

WEEK13

Tuesday

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