Team:Tsinghua/Notebook

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WEEK 1 (5.18 – 5.24)
5.18 HUANG, MAO, ZHOU, XIONG
-Transformation of BBa_I15009 and BBa_K592018 into DH5α.
5.19 HUANG
-Incubation of colonies of DH5α transformed with BBa_I15009 and BBa_K592018.
5.20 HUANG
-Preparation of BBa_I15009 and BBa_K592018.
5.24 HUANG, CUI, ZHOU
-Transformation of BBa_I15009 and BBa_K592018 into DH5α.

WEEK 2 (5.25 – 5.31)
5.25 HUANG, WANG, ZHOU
-Incubation of colonies of DH5α transformed with BBa_I15009 and BBa_K592018.
-Identification of BBa_K592018.

WEEK 3 (6.1-6.7)
6.2 ZHOU
-Transformation of BBa_K592018, BBa_I15008, BBa_I15009, BBa_R0082, BBa_M30011, BBa_K952003, BBa_K592002, BBa_K592029, BBa_K907000 and BBa_K1218011.
6.3 MAO, HUANG
-Transformation of BBa_K592029 failed.
-Incubation of colonies of DH5α transformed with BBa_K592018, BBa_I15008, BBa_I15009, BBa_R0082, BBa_M30011, BBa_K952003, BBa_K592002, BBa_K907000 and BBa_K1218011.
-Preparation of BBa_K592018, BBa_I15008, BBa_I15009, BBa_R0082, BBa_M30011, BBa_K952003, BBa_K592002, BBa_K907000 and BBa_K1218011.
-Identification of BBa_K592018, BBa_I15008, BBa_I15009, BBa_R0082, BBa_M30011, BBa_K952003, BBa_K592002, BBa_K907000 and BBa_K1218011.

1-9 on this figure represented BBa_K592018, BBa_I15008, BBa_I15009, BBa_R0082, BBa_M30011, BBa_K952003, BBa_K592002, BBa_K907000 and BBa_K1218011. A and a, B and b were from the same colony. Uppercase represented digested plasmid, lowercase represented undigested plasmid.
1-3 and 6-9 were successful.
6.4 HUANG
-Identification of BBa_R0082 and BBa_M30011.
6.7 HUANG
-PCR amplification of T1 and TE terminator, YF1+FixJ, mRFP, and FixK2 promoter.

WEEK 4 (6.8 – 6.14)
6.9 HUANG, CUI
-Gel extraction of T1 and TE terminator, YF1+FixJ, mRFP, and FixK2 promoter.

-Overlap PCR of Blue Light System I and II.
-Gel extraction of Blue Light System II.

-Ligation of Blue Light System II.
6.13 HUANG
-Overlap PCR of Blue Light System I. Failed again.

WEEK 5 (6.15 – 6.21)
Final exam

WEEK 6 (6.22 – 6.28)
6.26 XIONG, WANG
-PCR amplification of Blue Light System I components.
-Enzymatic digestion of the components.
-Molecular cloning failed again.
WEEK 7 (6.29 – 7.5)
7.3 CUI
-PCR amplification of Blue Light System I components.
-Gel extraction.

7.4 CUI
-Enzymatic digestion of YF1+FixJ, T1 and TE terminator, and promoter 104.

WEEK 8 (7.6 – 7.12)
7.6 CUI
-PCR amplification of Blue Light System I components, again.
7.7 MAO
-PCR amplification of T1 and TEterminator flanked by loxP. Failed.
-Transformation of HCKan_O into DH5α.
7.8 CUI
-PCR amplification of Blue Light System I components, AGAIN.
-PCR product purification.
-Enzymatic digestion of the products.
-Ligation of the digestion products.
-PCR amplification of the ligation product. Failed.
7.9 CUI
-Another round of the same cloning process.
7.10 CUI, MAO, WU
-Transformation of HCKan_O, pSB1C3, BBa_C0051, pKD46, pKD13, and pCP20 into DH5α.
-Culture of Trans1-T1 from Chen Lab.
-Gel extraction of digestion products.
-Ligation of digestion products.
7.11 MAO, ZHOU, WU, CUI
-No colony of DH5α transformed with HCKan_O, pSB1C3, pKD46, pKD13, and pCP20 present.
-Incubation of colonies of DH5α transformed with BBa_C0051.
-Transformation of HCKan_O, pSB1C3, pKD46, pKD13, and pCP20 into DH5α
-Preparation of competent cells from Trans1-T1.
-PCR amplification of ligation product. Failed again.
7.12 WU
-Incubation of colonies of DH5α transformed with HCKan_O, pSB1C3, pKD46, pKD13, and pCP20.

WEEK 9 (7.13 – 7.19)
7.13 CUI, WU
-PCR amplification of T1 and TE terminator.
-Enzymatic digestion of Blue Light System I components.
-Gel Extraction of digestion products.
-Preparation of pSB1C3, BBa_C0051, pKD46, pKD13, and pCP20.
7.14 CUI
-Ligation of Blue Light System I components.
7.16 CUI, WU
-PCR amplification of Blue and Red Light System components.
-Sequencing of pKD46, pKD13, BBa_C0051.
-Incubation of colonies of DH5α transformed with pKD46 and pKD13.
7.17 CUI, WU
-Gel extraction of PCR products of Blue and Red Light System components.
-PCR amplification of T1 and TE terminator for Red Light System and promoter 104 for Red Light System.
7.18 CXY
-Gel extraction of T1 and TE terminator for Red Light System and promoter 104.

BLT: T1 and TE terminator for Blue Light System. RLBB: Backbone for Red Light System. BLBB: Backbone for Blue Light System.
-Seamless cloning of Blue Light System (PLS001) and Red Light System I (PLS002).
-Transformation of DH5α with PLS001 and PLS002.
7.19 WU
-Incubation of DH5α transformed with PLS001, PLS002, pKD13, pKD46, and pCP20.

WEEK 10 (7.20 – 7.26)
7.20 CUI, WU
-PCR amplification of ho1 and pcyA.
-Transformation of pKD46 into Trans1-T1 and DH5α.
-Seamless cloning of Red Light System I (PLS003).
-Transformation of DH10B with PLS003.
7.21 WU
-Preparation of PLS001, pKD13, pKD46, and pCP20.
-Incubation of DH5α and Trans1-T1 transformed with pKD46. Add L-arabinose into the suspension.
-Gel extraction of Kana Cassette.
7.22 WU, CUI
-Electro-transformation of Kana Cassette into DH5α.
-Transformation of PLS003 into DH10B.
-Seamless cloning of Red Light System I (PLS004).
-PCR amplification of components of Red Light System.
-Gel extraction of PCR products.

7.24 MAO, WU, CUI
-Incubation of △Envz Trans1-T1 treated with and without arabinose.
-Sequencing of PLS001 and PLS004.
7.25 WU
-Incubation of EnvZ 1.
-PCR identification of EnvZ1 and negative control.

WEEK 11 (7.27 – 8.2)
7.28 WU, CUI
-Incubation of △Envz DH5α.
-A new round of PCR amplification of components of Blue and Red Light System.
-Seamless cloning of Blue Light System (PLS005) and Red Light System I (PLS006).
-Transformation of PLS005 and PLS006 into DH5α.
7.31 WU, ZHOU
-PCR identification of △Envz DH5α. Failed.
-Incubation colonies of DH5α transformed with pKD13 and pCP20.
8.1 ZHOU
-PCR identification of △Envz DH5α.

-Preparation of pKD13.
8.2 ZHOU
-Identification of pKD13.
Expected fragment length: 1812bp, 892bp, 479bp and 251bp.

-Preparation of pCP20.

WEEK 12 (8.3 – 8.9)
8.3 WU, MAO, ZHOU
-Transformation of pKD46 into DH5α.
-Identification of pKD13 and pCP20.

8.4 WU, ZHOU
-Incubation of colonies of DH5α transformed with pKD46.
-Identification of pKD13. Failed.
-Gel extraction of Kan2.
8.5 WU, XIONG, ZHOU
-Electro-transformation of Kan2 into RecA+ DH5α.
8.6 WU, ZHOU.
-Incubation of colonies of △Envz DH5α.
-Transformation of pKD13 into DH5α.
8.7 XIAO, ZHOU.
-Colony PCR of △Envz DH5α.

-Transformation of plv-dCas9-sgRNA.
8.8 WU, ZHOU
-Nest PCR △Envz DH5α and negative control.
-Incubation of colonies of DH5α transformed with pKD13.
8.9 ZHOU, XIAO
-Nest PCR of △Envz DH5α and negative control.

-Transformation of BBa_K747041 and BBa_K592029

WEEK 13 (8.10 – 8.16)
8.10 XIAO, ZHOU
-Incubation of colonies of △Envz DH5α and DH5α transformed with BBa_K747041, BBa_K592029, PLS005 and PLS006.
8.11 XIAO, ZHOU
-Preparation of BBa_K747041, BBa_K592029, PLS005 and PLS006.
-Sequencing of PLS005 and PLS006.
-Incubation of △Envz DH5α and DH5α transformed with plv-dCas9-sgRNA.
8.12 ZHOU, HUANG
-Incubation of colonies of △Envz DH5α.
-Preparation of plv-dCas9-sgRNA.
-Sequencing of BBa_K747041, BBa_K592029, plv-dCas9-sgRNA and PLS005.
-Ligation of Vika, Sore, Vore, Dre and FLPE onto pSB1C3.
-Transformation of ligation products.
8.13 ZHOU, HUANG
-PCR amplification of components of Red Light System.
-Identification of PLS005.
-Identification of △Envz DH5α..

-PCR amplification of components of Red Light System I.
-Seamless cloning control experiment.
-Transformation of BBa_K731520.
8.14 WU
-Incubation of colonies of △Envz DH5α.
8.15 MAO
-PCR amplification of FRT-cassette, Rep ori + Rep101, FLP and RecA.
-Enzymatic digestion of the fragments.
-Seamless cloning of the fragments onto pKB1C3.
-Transformation of the reaction products.
8.16 MAO
-Preparation of PLS005 and PLS006.
-Identification of PLS005 and PLS006. All were false positive.

WEEK 14 (8.17 – 8.23)
8.17 MAO
-Preparation of BBa_K1797011, PLS007 (FLP) and PLS008 (FRT).
-PCR amplification of Reps101.
-Identification of pSB1C3, PLS007, PLS008 and BBa_K1797011.

8.18 MAO, WU, CUI
-Enzymatic digestion of pSB1C3.
-Seamless cloning of Red Light System I (PLS009).
-Transformation of PLS009.s
-Electro-transformation of pKD46.
8.21 ZHOU, MAO
-Seamless cloning of Blue Light System (PLS010).
-PCR amplification of FRT-cassette, Rep Ori + Rep101, FLP and RecAs.
8.22 WU, HUANG
-Anneal of PLS011.
-Transformation of PLS011.

WEEK 15 (8.24 – 8.30)
8.24 XIAO
-Incubation of colonies of DH5α transformed of pKD46.
8.25 XIAO
-Preparation of pKD46.
-ara-ccdb construction
8.27 HUANG
-ara-ccdb testing, failed
8.27 HUANG
-iPTG-ccdb construction

WEEK 16 (8.31 – 9.6)
9.1 HUANG
-iPTG-ccdb qualitatively testing
9.2 WU
-Blue system construction
-Insert promoter 104 to 2-21N
9.3 WU
-Infusion cloning:
dCas9-linker1-Flp, dCas9-linker2-Flp, dCas9-linker1-Bxb1 and dCas9-linker2-Bxb1
9.6 HUANG
-Identification of PLS012.
-parts construction

WEEK 17 (9.7 – 9.13)
-Measurement of OD600 after treatment of iPTG gradients.

iPTG concentration/nmol·μl-1	0.00	1.40	2.80	4.20	5.60	6.99	8.39
OD600	1.71	1.69	1.69	1.79	1.57	1.61	1.56
iPTG concentration/nmol·μl-1	9.79	11.19	12.59	13.99	15.39	16.79	18.18
OD600	1.51	1.39	1.30	1.11	1.18	1.00	0.92
iPTG concentration/nmol·μl-1	19.58	20.98	22.38	23.78	25.18	26.58	27.98
OD600	0.83	0.94	0.65	0.59	0.51	0.35	0.30
iPTG concentration/nmol·μl-1	29.37	30.77	32.17	33.57	34.97	36.37	37.77
OD600	0.14	0.11	0.07	0.05	0.04	0.04	0.02

-sgRNA library construction, failed.
9.8 ZHOU, WU
-Trans1T1 pKD46 transformation
9.9 ZHOU, WU
-Pick Trans1T1-pKD46, DH5-pKD46 and culture at 30
-Kana cassette PCR, gel electrophoresis and gel extraction
9.10 ZHOU, WU
-Add L-arabinose to induce pKD46 express the recombination system, make electroporation-competent cells and electro-transform Kana cassette
-Parts construction by overlap extension PCR and enzyme digestion-ligation: RecA, gam-bet, exo, kan-cassette, Rep-ori
9.12 ZHOU
-Pick colonies and PCR with primer33 and primer34. Failed.
-Colony PCR with primer34 and K1R. Failed.
-Electro-transform pKD46 into DH5 

9.13 ZHOU
-Pick more colonies transformed kana cassette and do PCR. Failed.
-PCR kana cassette using new primers and purify.
-Pick DH5-pKD46 colonies and culture

WEEK 18 (9.14 – 9.20)
-sgRNA library construction, failed.

9.14 MAO, CUI, ZHOU
- Measurement of standard curve of OD value against relative concentration of bacteria

Bacteria/Dilution factor	1	2	3	4	5	6	7	8	9	10
	2.00	1.34	0.99	0.78	0.63	0.54	0.53	0.39	0.36	0.31
	2.00	1.37	1.00	0.77	0.63	0.54	0.49	0.41	0.36	0.32
	1.10	0.61	0.43	0.32	0.27	0.24	0.18	0.17	0.15	0.15

-Electro-transform kana cassette into DH5-pKD46

9.15 WANG, MAO, HUANG

9.18 ZHOU
-Pick DH5-pKD46-kana cassette colonies and PCR with primer33 and primer34.

 

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