Team:ZJU-China/Notebook/August

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Labnote

Biobrick group | Biofilm group

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

July

In this month we......
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August

In this month we......
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September

In this month we......
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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.

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

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

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.

1st August

Freeze slicing of about 50μm. Observe under natural light microscope and can see red florescence. The thickness is about 130μm

4th August

Repeat the last experiment with silicone tube. 6rpm/s results in a flowing speed of 68ml/day

6th August

Similar to the last time. Did not use freeze slicing.

15th August

Cultured E.coli in 5ml LB for 12h.

16th August

Substituting a part of silicone tube with glass tube in silicone tube biofilm formation sets. Culture in 37℃

17th August

Add 50ml LB and culture with circular culture.

18th August

12.00 found one free-flow pump stopped. Cooled for 15 minutes and turned on again. Could be over heated. Can see some bacteria on the bottom of the vessel and tube. When the pump started again, the bacteria was washed away.

19th August

Can observe obvious white biofilm where the silicone tube joins the tube but cannot see red florescence, suspect contamination. No biofilm is observed on the glass tube under microscope.

21st, August

Biofilm formation with large test tubes. Inoculate with 1% e.coli. Rubber tubes are attached to air pump with filter between air pump and the tube to prevent entrance of germs. Two sets use MSM +glucose medium and two sets use LB.
Retry with glass tube biofilm formation set.

24th, August

Terminate biofilm formation, the glass slide left in the drawer to dry. Can see obvious rod like structure under 400 magnification. (spheres in MSM+glucose culture) No red florescence is seen. Most of the surface is covered by single layer cells but some part of it has thick bump like structure.
No biofilm observed on the glass tube.