Team:ZJU-China/Notebook/September

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Labnote

Biobrick group | Biofilm group

This group...........

May&June

In the two months we discussed some new ideas and finally decided our project
>> Click to see our brainstorm

July

In this month we......
>>Click to see

August

In this month we......
>>Click to see

September

In this month we......
>>Click to see

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.

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)

Week11

Day

Note

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.

Week12

Day

Note

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.

3rd Sep

Directly use DH5α fdhF+RFP colony to inoculate silicone tube set with 5ml LB. rest for 12h for biofilm attachment.

4th Sep

Add 50mlLB+Amp and start circulation.

6th Sep

Biofilm with direct inoculation is optimum. Expressed red florescence but only one small fraction of the biofilm remained after cutting off the part of the tube.

12th Sep

4 silicone tube sets with biofilm attachement time of 3h, 5h, 7.5h and 10h; 4 bubbling sets.

15th Sep

The silicone tube set with 3h of attachment achieves optimum biofilm formation. Bubbling sets glass slides are froze in -80℃ for later observation with laser confocal. Biofilm formed on glass slide is about 10μm thick.

16th Sep

New sets with silicone tube, a mixture of RFP, CFP and YFP expressing e.coli. Attachment 3h.

17th Sep

Glass slide with bubbling set with the same mixture of e.coli.
Place two filter film on solid culture medium and placed the mixture of e.coli on the filter to form gas-solid interface biofilm.

20th Sep

Observation of glass slide and gas-solid biofilm under laser confocal. Glass slide are observed directly and gas-solid biofilm is first pressed by a covering slide so that it would stick to it and peeled off from the filter for observation.
Biofilm on glass slide is about 8.6μm thick. Biofilm formed on gas-solid interface is too concentrated in bacteria and culture medium that the florescence of too strong to see any clear structure.