Team:WHU-China/Notebook/Daytoday

4/14~4/15, preparing materials: such as DH5α, pDsRed -Express Vector, amp, kana, TB, 0.85%NaCl, 0.1MCaCl2, 0.1MMg2+
4/24, successfully obtained pDsRed-Express Vector plasmid
4/26~4/29, transform pDsRed-Express Vector plasmid into DH5αcompetent cell in order to test our experiment skills
5/1~5/10, vocation

5/11~5/26, use enzyme SpelⅠto digest pDsRed-Express Vector plasmid
5/27, use XbalⅠto digest pDsRed-Express Vector plasmid
5/28, use EcoRⅠto digrst pDsRed-Express Vector plasmid
5/29~6/1, use Pst1 and EcoRⅠto digest pDsRed-Express Vector
6/2~6/15, produce the competent cell, use T4 ligase to join DNA fragments, and conduct the transformation..During those days, we modified the procedures of competent cell production and successfully boomed the results of transformation to 10^7
6/16~7/7, too busy with the school stuff

7/8~7/9, pick up the procedures of transformation after the break. At the meantime, use PstI and EcoRI to digest the pDsRed-Express Vector, conduct the gel extraction and join the DNA fragments overnight
7/10, transform the products of ligation
7/11, tranform the plasmid of pSB1A3, pSB1C3 and biobrick 15NI, 6AI
7/12, re-transform pSB1C3, biobricks 15NI, 6AI
7/13, extract the plasmid of 15NI, 6AI, use EcoRI and SpeI to digest the 15NI plasmid, use EcoRI and XbalI to digest the plasmid of 6AI
7/14, re-transform the pSB1A3 and the biobricks of 15NI 6AI due to the mistakenly omitting steps of culturing E. Coli with these plasmids

7/15~7/17, transform the plasmids of 23LⅠ,6HⅢ，17PⅠ,2KⅠ,16MⅣ，2MⅠ, 13JⅡ，20FⅠ,8HⅡ，14HⅠ,14KⅠ,20KⅠ,4AⅠ,6IⅠ,14JⅠ,12AⅠ,4MⅠ,2IⅠ,4OⅠ At the meantime, digest the 4AⅠ, 4OⅠ, 4MⅠ with EcoRI and SpelⅠ, 23LⅠ with EcoRI and XbalⅠ, then conducting ligation digest the 6HⅢ and 17PⅠwith EcoRⅠ and SpelⅠ, with an unexpected 3-band result, owing to the higher than normal plasmid concentration.
7/18, transform the biobricks of 8HⅡ,14KⅠ,20 KⅠ, 6IⅠ, 12AⅠ, gaining the ligation products of 23LⅠ-4AⅠ，23LⅠ- 4OⅠ，23LⅠ- 4MⅠ23LⅠ-6HⅢ, re-digest 6HⅢ with EcoRⅠ and SpelⅠ.
7/19, transform the biobricks of 16MⅣ，20FⅠ，14HⅠ，14JⅠ，13JⅡ, use gal extract to gain the product.
7/20, use the EcoRⅠand SpelⅠto digest 4AⅠ, 4OⅠ, 4MⅠ,use EcoRⅠand XbalⅠto digest 23L，in the meantime we use enzyme digest to test our product 4AⅠ, 4OⅠ, 4MⅠ，20 KⅠ, 6IⅠ, 12AⅠ，gain our ligation product 23LⅠ- 4MⅠ，23LⅠ-6HⅢ

7/21, complete the ligation of 23LⅠ-4AⅠ，23LⅠ- 4OⅠ，23LⅠ- 4MⅠ，23LⅠ- 14KⅠ
7/22, transform the biobricks of 6A and 15N，complete the ligation of 20 FⅠ-2MⅠ，13JⅡ-2M.
7/23~7/28, Focused on the ligation of biobricks . Till 7/28 we have successfully ligate the 23LⅠ- 4MⅠ，23LⅠ- 4OⅠ，23LⅠ-14KⅠ，20FⅠ-2MⅠ，13JⅡ-2MⅠ，23LⅠ-14JⅠ，23LⅠ-6HⅢ，4AⅠ-23LⅠ-2MⅠ，20 FⅠ-2MⅠ-23LⅠ，and 17PⅠ-23LⅠ
7/29, ligated the 14JⅠ-23LⅠ-2MⅠ，4OⅠ-23LⅠ-2IⅠ，14HⅠ-2MⅠ

7/30, ligated 8HⅡ-2MⅠ，4AⅠ-23LⅠ-2MⅠ-20KⅠ，15NⅠ-23LⅠ-6HⅢ，2KⅠ-16MⅣ，15NⅠ-23LⅠ-17PⅠ，2MⅠ-13JⅡ-2MⅠ-20 FⅠ-23LⅠ
7/31, ligate 2MⅠ-23LⅠ- 4MⅠ，4OⅠ-23LⅠ-2IⅠ- 6IⅠ，14JⅠ-23LⅠ-2MⅠ- 6IⅠ
8/1~8/2, ligate RLS5+6(2KⅠ-16MⅣ-2MⅠ-13JⅡ-2MⅠ-20 FⅠ-23LⅠ) RLS1+6(2KⅠ-16MⅣ-2MⅠ-13JⅡ-2MⅠ-20 FⅠ-23LⅠ)
8/3, the ligation of 4AⅠ-23LⅠ-2MⅠ-20KⅠ,8HⅡ-2MⅠfailed，but we successfully gained the 4OⅠ-23LⅠ-2IⅠ- 6IⅠ，2MⅠ-23LⅠ- 4MⅠ，ligated the 2MⅠ-23LⅠ- 4MⅠ-12AⅠ

8/4~8/5, re-ligated the 23LⅠ-6HⅢ，17PⅠ-23LⅠ，4AⅠ-23LⅠ-2MⅠ-20KⅠ，test RLS1+6(2KⅠ-16MⅣ-2MⅠ-13JⅡ-2MⅠ-20 FⅠ-23LⅠ)，transformed new biobricks 19CⅣ，15OⅣ，19IⅣ，6JⅢ,6LⅢ，6NⅢ，16EⅠ，8LⅡ，12LⅠ，20ⅠⅠ,6NⅠ
8/6, re-ligated 14JⅠ-23LⅠ-2MⅠ- 6IⅠ，ligated 14HⅠ-2MⅠ，6JⅢ-23LⅠ，6LⅢ-23LⅠ，15NⅠ- RLS1+6，19CⅣ-23LⅠ，15OⅣ-23LⅠ，19IⅣ-23LⅠ，23LⅠ-14KⅠ，10EⅣ-23LⅠ，15MⅣ-23LⅠ，17AⅣ-23LⅠ
8/7~8/10, ligated14JⅠ-23LⅠ-2MⅠ-6IⅠ，2MⅠ-14HⅠ-2MⅠ，6JⅢ-23LⅠ，6LⅢ-23LⅠ，15NⅠ-RLS1+6，19CⅣ-23LⅠ，15OⅣ-23LⅠ，19IⅣ-23LⅠ，15MⅣ-23LⅠ，17AⅣ-23LⅠ
8/11~8/12, ligated 8HⅡ-2MⅠ-14HⅠ-2MⅠ，2MⅠ-19CⅣ-23LⅠ，2MⅠ-15MⅣ-23LⅠ，2MⅠ-15OⅣ-23LⅠ，use enzyme to verify the 6JⅢ-23LⅠ，6LⅢ-23LⅠ，17AⅣ-23LⅠ
8/13~8/14, re-ligate 2MⅠ-19CⅣ-23LⅠ，2MⅠ-15MⅣ-23LⅠ，2MⅠ-15OⅣ-23LⅠ，ligate 2MⅠ-17AⅣ-23LⅠ，2MⅠ-6JⅢ-23LⅠ，2MⅠ-6LⅢ-23LⅠ
8/15, re-ligate 8HⅡ-2MⅠ-14HⅠ-2MⅠ，14JⅠ-23LⅠ-2MⅠ-6IⅠ，23LⅠ-14KⅠ，use PCR to amplify the 2MⅠ-19CⅣ-23LⅠ，2MⅠ-15MⅣ-23LⅠ，2MⅠ-15OⅣ-23LⅠ，2MⅠ-17AⅣ-23LⅠ

8/16, ligate 16EⅠ-2MⅠ-6LⅢ-23LⅠ，16EⅠ-15OⅣ，16EⅠ-17AⅣ，16EⅠ-19CⅣ，16EⅠ-15MⅣ
8/17~8/18, ligate 4EⅠ-23LⅠ，RLS1+6-16EⅠ-15OⅣ，RLS1+6-16EⅠ-6LⅢ，RLS1+6-16EⅠ-17AⅣ，RLS1+6-16EⅠ-19CⅣ，RLS1+6-19CⅣ，16MⅣ-2KⅠ
8/19~8/20, ligate 8HⅡ-2MⅠ-14HⅠ-2MⅠ-23LⅠ-14KⅠ，14JⅠ-23LⅠ-2MⅠ-6IⅠ-4AⅠ-23LⅠ-2MⅠ-20KⅠ，15NⅠ-RLS，16EⅠ-2MⅠ-19CⅣ-23LⅠ- pSB1C3, 16MⅣ- RLS6
8/21~8/22, re-ligate 4OⅠ-23LⅠ-2IⅠ- 6IⅠ，14JⅠ-23LⅠ-2MⅠ-6IⅠ，23LⅠ-14KⅠ，8HⅡ-2MⅠ-14HⅠ-2MⅠ，16EⅠ-2MⅠ-19CⅣ-23LⅠ- pSB1C3，16MⅣ- RLS6
8/23, ligate 14JⅠ-23LⅠ-2MⅠ-6IⅠ-4AⅠ-23LⅠ-2MⅠ-20KⅠ，4OⅠ-23LⅠ-2IⅠ- 6IⅠ，8HⅡ-2MⅠ-14HⅠ-2MⅠ-23LⅠ-14KⅠ，16EⅠ-2MⅠ-19CⅣ-23LⅠ-pSB1C3，16EⅠ-15MⅣ, 16EⅠ-15OⅣ, 16EⅠ-17AⅣ, 16EⅠ-6LⅢ
8/24~8/25, 16EⅠ-15MⅣ-pSB1C3, 16EⅠ-15OⅣ-pSB1C3, 16EⅠ-17AⅣ-pSB1C3, 16EⅠ-6LⅢ-pSB1C3，16EⅠ-2MⅠ-19CⅣ-23LⅠ-pSB1C3, RLS-18OⅠ, RLS-18MⅠ, RLS-2GⅡ,k360123-k360126

8/26, extract plasmid: 009-13,009-3. Enzyme digest 009-13,009- 3. Result shows that it is not cut completely .PCR 20 + 2 positive control.Transformation: 2 M-13 J-2 M-20 F, PSB, 2 K-16 MIV, CHL1-CHL3 (DH5 α and TrpR defects strains)
8/27, extract plasmid: 8H, 2M-14 H-2M. enzyme digest 009-3, 8H, 2 M-14 H-2 M. Ligate 009-2-3.Identification of the transformation of PCR 8.26 ligation results. Cut CHL1 religate CHL1-CHL3, CHL1-CHL4.Enzyme cut PCB, 23 L, RLS1.Transformation: CHL1-CHL3 (DH5 α and TrpR defects plant), CHL1-CHL4 (DH5 α and TrpR defects plant), 009-2-3, controls.
8/28, repeat 27th enzymes digest,ligated 009-2-3. Transformation: 18 M-RLS1, 18 O-RLS1, 2 G Ⅱ-RLS1, PSB-23 L, 009-2-3, CHL1-CHL3 (TrpR defects plant), CHL1-CHL4 (TrpR defects strains) controls. Reserve RLSF-2, PSB-1.
8/29, PCR 2 M-19 C, Enzyme cut 2 M -14 H-2 M, 14 K; transform 118,122,2M-18F,009-5,009-13,control.
8/30, extract plasmid 18 O I, 18 M I, 2 G II-RLS1 and PSB-23 L /11 tube. PCR 2 M-18 F, and identified positive cloning, the result is positive.Enzyme cut 009-2-3, 009-1-6, 2 M-14 H-2 M. Cloning-5, 009 PCR positive. Ligate 14 K-23 L, 2 M-14 H-2 M-8 H.Transformation: 18 M-RLS-2-23 L, 18 M-RLS-3-23 L, 18 M-RLS-5-23 L, 18 M-RLS-6-23 L, 2 G-RLS-23 L-1, 2 G-RLS-23 L-4 and 16 E-PCB-23 L-4, 2 M-14 H-2 M-8 H, 14 K-23 L, controls.Reserve: 009-2-3-(4), 009-2-3-6, PCB-17, PCB-23 L-5, 2 G-RLS1-6, PCB-23 L-4, 18 M-RLS1-2, PCB-23 L-6, 2 G-RLS1-4.
8/31, PCR screening 18 O-RLS, 18 M- RLS RLS, 2 GII-RLS, PSB-23 L. PCR 2 M-15 M-23 L.Extract plasmid 009-5. enzyme cut 009-5 . ligated 14 H-2 M.Transformation: Plasmid 118, Plasmid 122, 14 H
9/1, extract plasmid 18 O, 18 M- RLS RLS, 2 GII-RLS, PSB-23 L. PCR 2 M-15 M-23 L,. PCR 2 M-17 A-23 L, 2 M-15 O-23 L, 2 M-15 M-23 L, 2 M-18 F. Enzyme cut and identificaty.Extract plasmid 009-13 (1-6), 009-14 (1-6), identify of the result of enzyme cut , ligate.Reserve: 009-14-1, 009-14-5, 009-14-6, 2 M-18 F-(1), 2 M-18 F-(2).Transformation: 009-13, 14 H-2 M, CHL1-3 (TrpR defects plant), controls.

9/2, enzyme cut 2 G-RLS, 18 O-RLS, ligate 23L; PCB-23 L, ligate 16E.Transformation: CHL1-4 (TrpR defects plant), 118, 122,, and the Plasmid Plasmid PCB-23 L, 18 O-RLS1-23 L, controls.
9/3, enzyme cut 2 G-RLS, 2 M-13 J, 18 O-RLS, 2 M-20 F, 2 M-13 J-23 L, 2 K-16 M, 16 M IV, 2 M-18 F. Extract plasmid 009-3.Transformation: Plasmid 118, 122, 009-Plasmid 2-3-4, 009-2-3-6, 009-5-10, controls.

9/4, extract plasmid 2 M-14 H-2 M, 14 H-2 M, enzyme cut ligation.extract plasmid PCB, 16 E, 2 M-13 J, 2 K, 2 M, 2 M-20 F, RLS, 2 M-17 A-23 L, 2 M-15 M-23 L, 2 M-19 C-23 L, 2 M-15 O-23 L, 2 K-16 M, RLS1-6. Enzyme cut identification.
9/5, enzyme digeat14 H, 18 O-RLS-23 L, PCB-23 L, electrophoresis, recycle.Transformation: CHL1-3 (TrpR defects plant), CHL1-4 (TrpR defects plant), 16 M Ⅳ, 20 F, 14 H, 17 A,19I、19C、15O、15M、control。
9/6, extract plasmid 2 M-14 H-2 M, and ligate.Enzyme 2 M-13 J. Extract plasmid 20 F I. Transformation: 14 H-2 M, controls.
9/7, enzyme cut 2 M I. Extract plasmid 2 M-15 O, 2 M-15 M, 2 M-19 C, 2 M-19 I, 2 M-13 J,2 M-17 , electrophoresis, enzyme cut identification. Extract plasmid 009-14. Transformation: Plasmid 118, pLPCB, Plasmid 118 & pLPCB co-transformation, 2 M since connection, 2 M-13 J Ⅱ, controls.Colonies PCR identification of 18 F.
9/8, ligate 2 K-13 J. Enzyme cut 009-14, ligate 14 K-23 L, 2 M-14 H-2 M. Transformation: pLPCB, 2 M. Ligate 16 M Ⅳ, 2 M-15 O, 2 M-13 J, 2 M-15 M, 2 M-17 A, 2 M-19 C, 2 M-19 I, 14 K-23 L, 2 M-14 H, controls.
9/9, extract plasmid 009-14, 8 H, RFP. Ligate 2 K-20 F. Transformation: 2 K-13 J, 2 K , 14 H-2 M, 14 K-23 L, 009-13, controls.
9/10, enzyme cut 2 K- 20 F, 2 K, PCR detection. Enzyme cut 2 M , 8 H, 15 M, 099-2-3, ligation.
9/11, extract plasmid15tubes, Enzyme cut identification. PCR 2M-13J, identification. enzyme cut14K-23L、8H、009-5. transformation：pLPCB、controls.
9/12, enzyme cut2M I、8H. extract plasmid009-13、15M-23L, enzyme cutligate14H-2M.
9/13, enzyme cut13J, phosphorylation2M、2K；enzyme cut18F. detectionCHL1-3、CHL1-4. ligate2Mself-ligate、2Kself-ligate、2K-13J、2K-16M、18F-2K-17A、2K-15M. colony PCR 15M-23L. enzyme cut009-2、009-3、009-4. Colony PCR identification transformation products. Blue Light Sensor test.

9/14, enzyme cut009-2、009-3、009-4、17A IV, recycle, ligate009-2+C、009-3+C、009-4+C、009-2-3+009-4、009-2-3self-ligate.
ligate2K、2M, enzyme cut detection.
Colony PCR identification transformation products.
transformation：009-2、009-3、009-4、009-2-3、009-2-3-4、2M-14H-2M、009-13、2M-17A-right、2M-17A-left、18F-2K-17A、2Kself-ligate、2Mself-ligate、16MⅣ、controls.
9/15, enzyme cut2M-13J、2K-20F、17A. ligate2M-13J-2K-20F、18F-2K-15O、GRE+118、2K-15O、2K-20F、13J-20F.
enzyme cut14H-2M, 2M-14H-2M, colony PCR. ligate009-2-3+009-4.
Blue Light Sensor test.
9/16, extract plasmid 009 2-3-4, 009,-13, 14 H, enzyme cut and identify. Colonies PCR. Ligate 4 O-23 L-2 I + 6 I.
Transformation: pJT122, GRE-118, 18 F-2 M-15 O, 2 M-17 A-right, 2 M-17 A-left, 2 M-13 J-2 M-20 F, 009-2, 009-3, 009-4, 15 M-23 L, 009-5, 14 H-2 M, controls.

9/17, enzyme 2 K-cut 20 F, 2 M-15 O, 18 F-2 M-15 O, 13 J-20 F, 18 F-2 M-15 O.
Mention plasmid 009-2-3-4, enzyme cut detection. Connection GRE--118, 009-2-3-4.
Colonies PCR product identification transformation.
PCR 2 M-18 F, PCR Plasmid 122.009 2-3-4
9/18, PCR detection 18 F-15 O. Enzyme cut 16 M IV.
9/19, link 2 K-16 M, 2 M-13 J-20 F, 18 F-15 O.6 I, 23 enzyme cut L, 009-2, 009-3, 009-4.
PCR 2 M-18 F.Transformation: 18 F-2 M-15 O, 2 M-16 M Ⅳ, 13 J-20 K, Plasmid 122, controls
9/20, extract plasmid: 2 M-18 F,and122.Colonies PCR product identification .PCR 2 M-18 F, PCR Plasmid 122.
9/21, PCR 18 F-15 O. Enzyme cut 15 O 14 H, 6 I, 23 L, 2 M.Colonies PCR identification .
PCR 2 M-18 F, PCR Plasmid 122.
9/22, extract plasmid 4 O-23 L-2 I, enzyme cut 4 O-23 L-2 I, 2 M, 6 I. PCR 18 O-23 L. Ligate 14 H + 2 M.

9/23, PCR JT112 , Cph8, 2 M-18 F. Cut CHL1 and CHL3. ligate CHL1-3.14 H, 6 I, 2 M, 4 O-23 L-2 I, 14 H-2 M.
9/24, PCR Cph8. Enzyme cut 2 M -15 O and 18 F, ligation.
9/25, PCR Cph8 , ligation18 F-15 O, enzyme cut 16 E, K30012 , J61002.
9/26, enzyme cut 2 M , 15 O-2 M.
9/27, PCR Cph8. PCR identify 16 E-13 J-20 F, 12 G, ligate 18 F-15 O-CHL1-4 (device of blue light sensor)
9/28, enzyme cut 2 M -18 F. Enzyme cut 12 G, CHL3. PCR 122.
9/29, G, enzyme cut 12 18 F. PCR Cph8.

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