June
6.8~6.15
1. transformation of BBa_B0015，BBa_CO261 to psb1AK3 is successful
2.Transformed pSB1T3,pSB1K3, pSB1C3,pSB1A3, BBa_I13504 and BBa_I13504.
4.the bacteria precipitate transformed with pSB1C3 and pSB1A3 were red after centrifugation, the degree of red decreased in this order: pSB1K32 (pinkred) - pSB1A3 (pink) - pSB1A3(tinge) - pSB1K3(yellow).
5. Chloramphenicol was insoluble，and it was tested to be useless using DH5α.
6.prepare A + ,K + ,T + ,C + plates and antibiotic store liquor.
7.Digested and ligated BBa_CO261, pSB1C3,then transformed them
9.Digestion of BBa_I13504, BBa_B0015, and proved that the transformation was successful
6.16~6.22
1.PCR to amplified 8B but failed
2. Chloramphenicol was tested to be effective
3.transformed pSB1T3, K2-8B BBa_CO261, BBa_B0015, BBa_I13504, pSB1K3(pSB1K3) and pSB1A3(pSB1A3)
4.plasmid extraction of F2622 + BBa_CO261
5.Digested F2622 + BBa_CO261 and pSB1T3 (EcoRⅠand PstⅠ)
6.ligated F2622 + BBa_CO261, BBa_B0015 and pSB1T3
6.23~6.29
1.strain conservation of F2622 + BBa_CO261, pSB1C3
2.prepared ddH2O
3.Added 1µl Dpn Ӏ into pSB1T3(pSB1T3) reaction system when digesting
July
6.30~7.6
1.Digested F2622 + BBa_CO261 and pSB1T3(EcoRⅠand PstⅠ)
2.ligated F2622 + BBa_CO261, BBa_I13504, and pSB1T3, but failed to transform
7.7~7.13
1.plated F2622 + BBa_CO261 + BBa_I13504(1.2.3) and F2622 + BBa_CO261 + BBa_B0015(1.2.3) which were ligated last week but failed.
2.cultured BBa_B0015 1 and 2
7.14~7.20
1.no much experiment
7.21~7.27
1. failed to amplify pSB1K3, pSB1A3, pSB1C3 through PCR
2. transformed K2-3G(RBS + Cre), K5-24B, K5-R0040 and 3G, 24B were successful
3.extracted plasmid of K2-8B, F2622 + BBa_CO261 and only K2-8B was successful
4.Liquid cultured 24B, 3G, R0040, 6O, 7F, 12C and F2622 + BBa_CO261 + BBa_I13504, F2622 + BBa_CO261 + BBa_B0015, 4F.
5. Successfully ligated F2622 + BBa_CO261 + BBa_I13504, F2622 + BBa_CO261 + BBa_B0015
RePCR by taq enzyme, we got experiences:
Taq is suitable for amplifying snippets of 2kb in size, and the reaction is fast
10X taq buffer with (NH4)2SO4 reduces nonspecification,
B0034M Mg2 + is suitable for taq
Avoid DNA pollution during template preparation
No pollution of raw material, it would be better to operate on the laminar
Negative control is necessary.
7.28~8.2
1.Recovered PCR product of 27th
2.ligated F2622 + BBa_CO261 + BBa_I13504, F2622 + BBa_CO261 + BBa_B0015 with pSB1C3 carrier, then did transformation and liquid culture
3.Liquid cultured R0040, 8B, 6O, F2622 + BBa_CO261, BBa_B0015
4.Replated F2622 + BBa_CO261 + BBa_I13504, F2622 + BBa_CO261 + BBa_B0015
5.Digested R0040, 6O, 24B
6.Ligated 6O with 24B, then did transformation
7.transformed 18O, 19C
August
8.3~8.10
1.ligated 6N + 19C + BBa_B0015, after failing to ligate 6N(T7, promoter) with 19C(reporter)
2.Liquid cultured 6O + 24B, and gel analysis of plasmid extraction and digestion products proved that the ligation was successful
3.Transformed K3- I746361, K3-K145215, K2-15J and 5J + 2C. K145215 and 15J turned out to be a failure. Liquid cultured the rest.
4.Digested 6O, 24B, pSB1C3, and ligated them overnight
8.11~8.17
1.transformed 5J + 2C，18O, 19C， I746361,K145215,15J，K5-J23032，F2622 + BBa_CO261
2.Extracted plasmid of K145215,15J,5J + 2C,F2622 + BBa_CO261
3.Digested K145215，6N + I13507，BBa_CO261，5J + 2C,F2622 + BBa_CO261，
4.Liquid cultured F2622，6N + 19C + BBa_B0015，J23032
5.Sent K145215, 6N + I13507, F2622, 6N + 19C(PSN1C3), 6N + 19C + BBa_B0015(PSB1C3) to biotech company to sequence them.
8.18~8.24
1.Ligated R0040 + J23032(Ptet + lock) ,B0034 + 2C , PO + LOX + R0040
2.Transformed B0034 + 6N,6O + 24B,F2622 + BBa_CO261, R0040 + J23032
3.Liquid cultured 5J + 2C, R0040 + J23032,6O + 24B,
4.Extracted plasmid of 5J + 2C，J23032, B0034（strong RBS）,14G + BBa_B0015, R0040 + J23032, 6O + 24B, 6N + B0034 and then did digestion.
5.Digested cre, po，6O + 24B, B0034,2C.
6.Sent 6O + 24B, 5J + 2C to sequence, however all turned out to be wrong, so we need to religate them.
7.Sent 5J + 2C + PSB1C3，14G + BBa_B0015 + PSB1C3, R0040 + J23032, 6N + B0034 to sequence
8.Amplified I746361 through PCR
8.25~8.31
1. Digested R0040 + J23032 and F2622 + BBa_B0015, B0034 + 2C, 6O + 24B
2.Designed primer of APO
3.Failed to PCR Cre + ssrA
4.Transformed B0034 + 2C, R0040 + PO,
5.Recultured 6O + 24B, F2622 + BBa_CO261
6.Single digested F2622 + BBa_B0015,R0040 + po, compared with previous single digested BBa_B0015, but failed.
7. Redigested BBa_B0015, then did ligation and transformation.
8.Sent to sequence: 14G + BBa_B0015, R0040 + J23032, 6O + 24B, R0040 + po, 6N + B0034, B0034 + 2C
9.Recovered BBa_B0015 and R0040 from conservation
September
9.1~9.7
1. succeeded in amplifying Cre + ssrA by PCR
2.Digested R0040, 6N + B0034, pSB1C3, pSB1K3, pSB1A3, pSB1T3, 6O + 24B, F2622 + BBa_CO261, Cre + BBa_B0015, 6N→BBa_B0015, B0034 + 14G + BBa_B0015, 4F + 6N, PCRed F2622 + BBa_B0015, F2622 + BBa_B0015 + R0040 + J23032, 6G, 24B
3.ligated Cre + BBa_B0015, F2622 + BBa_CO261(pSB1C3), B0034 + 14G + BBa_B0015(pSB1T3), 6N + B0034 + 14G + BBa_B0015, R0040 + J23032 + F2622 + BBa_B0015(pSB1T3), R0040 + po + I13507 (pSB1T3), RBS + Cre + ssrA + B0034 + 14G + BBa_B0015(pSB1T3) (C23 for short), RBS + Cre + ssrA (pSB1C3) (CreS for short), F2622 + BBa_B0015 (pSB1C3) (1223 for short), B0034 + 14G + BBa_B0015(pSB1C3), R0040 + BBa_I13504 (pSB1C3), I746361 + BBa_I13504 (pSB1C3), R0040 → BBa_B0015 (pSB1C3), 6G + 24B (pSB1C3), then transformed them.
4. Cultured 6O + 24B, B0034 + 2C
5.Digested pSB1C3, pSB1K3, pSB1A3, pSB1T3, 6O + 24B, F2622 + BBa_CO261
6.Transformed Cre + BBa_B0015, 18F + 4F, 4F + 6N, 6N + B0034 + 14G + BBa_B0015, pSB1A2, K56G, Plac, C23, 2012K1220, K33L, K3J04650, J04650
7.Transformed pSB1A2, K56G, Plac, C23, 2012K1220, K33L, K3J04650, J04650
8.Sent to sequence: B0034 + 14G + BBa_B0015, 6N → BBa_B0015, B0034 + 2C, 6O + 24B, 12C + BBa_B0015 → 7F, F2622 + BBa_B0015 → 1B0034
9.Did gel extraction of 6N + 19C + BBa_B0015, RBS + Cre + ssrA
9.8~9.14
1.Digested CreS, 1223, B0034 + 14G + BBa_B0015, 6N→BBa_B0015, I13507, pSB1C3, CreD, CreE, R0040 + po, R0040 + po + I13507, 6G + 3L
2.Liquid cultured R0040→BBa_B0015, 6G + 24B
3. Ligated 6G + 3L, R0040 + po + I13507, R0040 + J23032 + J04650 ,R0040 + po + I13507, CreS, C23
4.Sent to sequence: 1223, B0034→BBa_B0015, 6N→BBa_B0015, R0040→BBa_B0015, 6G + 3L, R0040 + po + I13507
5.Traditional assembly of Cre and C23
6.96-well plates experiment of R0040 + po
7.Cultured R0040 + BBa_I13504 and observed its fluorescence
8.Transformed R0040 + J23032 + J04650, 6G + 3L, R0040 + po + I13507
9.15~9.21
1.Miniprep：I746361 + I13507, 6G + 3L, R0040 + J23032 + J04650, R0040 + Po + I13507, I746361 + I13507, Cres + B0034 + 14G + BBa_B0015, I746361 + I13507 + R0040→BBa_B0015, K145215 + R0040 + J23032 + J04650, I746361 + I13507 + R0040 + J23032 + F2622 + BBa_B0015 + K145215, R0040 + po + I13507, 6g, I746361→BBa_B0015 + K145215, C23, I13521, K145215 + R0040 + J23032 + J04650
2.Digestion: I746361 + I13507, R0040 + J23032 + 12K, Cres, 5I + J23032 + J04650, Cre + B0034 + 14G + BBa_B0015, I746361 + I13507 + R0040→BBa_B0015,K145215 + R0040 + J23032 + J04650, R0040 + Po + I13507, R0040 + po + I13507, 6g, I746361→BBa_B0015 + K145215
3.Liquid culture: I746361 + I13507, Cres, I746361 + I13507 + R0040→BBa_B0015, Cre + B0034 + 14G + BBa_B0015, cres + Po + I13507, 6G, R0040 + J23032 + J04650, K145215 + R0040 + J23032 + J04650, R0040, K145215 + B0034 + J23032 + BBa_B0015, R0040 + Po + I13507
4. Prepared A + and T + plates
5. Ligated I746361→BBa_B0015 + K145215 and R0040 + I13507,then did transformation
6. Function test: K145215 + R0040 + J23032 + J04650, R0040 + J23032 + J04650, R0040 + J23032 + J04650 + K145215, I13521, I746361 + I13507, I746361 + I13507 + R0040→BBa_B0015, I746361 + 220 + R0040→BBa_B0015 + K145215, R0040 + Po + I13507
7. Gel extraction of R0040 + po + I13507 and 6g
8. Sent 6G + 3L, R0040 + J23032 + J04650 + K145215, I746361 + I13507 + R0040→BBa_B0015, I746361 + 220 + R0040→BBa_B0015 + K145215 to sequence
9.22-9.28
1.Resent I746361 + 220 + R0040→BBa_B0015 + K145215 to sequence
2.Prepare LB liquid culture and LB plates
3.Liquid cultured 6G + R0040 + PO + I13507, C23,B0034→BBa_B0015 and 3L, 6G→I13507, and then extracted plasmid
4.Digested I13521、C23,B0034 + 14G + BBa_B0015、3L, cre、R0040 + po + I13507 + 6G、6G→I13507、B0034→BBa_B0015、B0034 + 14G + BBa_B0015 + psb1c3
5.Gel extraction of B0034 + 14G + BBa_B0015、3L、B0034 + 14G + BBa_B0015 + psb1c3、cre、3L、6G→I13507 and I13507
6.Function test of I13521、 I746361→BBa_B0015、 I746361→K145215、 I746361 + I13507
7.PCRI13507
8.Ligated cre and B0034→BBa_B0015
9.Transformed c23、B0034→BBa_B0015 and 3L
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