Team:ZJU-China/Notebook

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Biobrick

brief introduction

Biofilm Formation

brief introduction

week1
In this week we........
week2
week3
week4
week5
week6
week7
week8
week9
week10
week11
week12
after week12

Week1

Day

Note

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

There is no data in first week

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

•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

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

•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

Day

Note

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

Day

Note

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

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Week6

Day

Note

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

Day

Note

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

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Week8

Day

Note

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

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Week9

Day

Note

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

Day

Note

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

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Data&protocol

Monday

WEEK11

Tuesday

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Data&protocol

Monday

WEEK12

Tuesday

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Data&protocol

Monday

WEEK13

Tuesday

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