Team:Osaka/week1

August 1 (Mon)

1. Preparation of LB agar plates (33 Amp, 18 Kan, 17 Cam).

August 2 (Tue)

1. Culture medium preparation
2. Competent cells preparation - Nojima Method
   • SOB medium (MgCl2 not yet added) -> stored at 4˚C
   • Preparation of glucose solution for making SOC medium.
   • Single-colony streaking of E. coli (strain DH5α) on 2 LB agar plates -> 37˚C incubation o/n
3. Transformation of Registry parts (See Table 1).

Note: 'ID' is an internal identifier used by wet work members to simplify labeling etc. In the case of iGEM distribution parts it usually refers to the plate location.

Table 1

ID	Part Name	Resistance	Description
1-9N	<bbpart>BBa_J22106</bbpart>	A	rec A (SOS) Promoter
1-2M	<bbpart>BBa_K118022</bbpart>	A	RBS
1-14K	<bbpart>BBa_B0040</bbpart>	A	green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP
1-12M	<bbpart>BBa_E0240</bbpart>	A	GFP generator
1-1D	<bbpart>BBa_R0010</bbpart>	A	promoter(lacI regulatd)
3-6H	<bbpart>BBa_K274100</bbpart>	A	CrtEBI with rbs
2-18H	<bbpart>BBa_K118014</bbpart>	A	rbs+crtE (geranylgeranyl pyrophosphate synthase)
2-16N	<bbpart>BBa_K118006</bbpart>	A	rbs+crtB (phytoene synthase)
2-18H	<bbpart>BBa_K118005</bbpart>	A	rbs+crtI (phytoene dehydrogenase)
1-17P	<bbpart>BBa_I742155</bbpart>	A	crtY (lycopene cyclase) with native rbs
1-1G	<bbpart>BBa_J04450</bbpart>	A	construction plasmid containing mRFP coding device
1-3A	<bbpart>BBa_J04450</bbpart>	C	construction plasmid containing mRFP coding device
1-5A	<bbpart>BBa_J04450</bbpart>	K	construction plasmid containing mRFP coding device

Tomorrow we shall prepare the remainder of the reagents and inoculate SOB with o/n-incubated E. coli.

August 3 (Wed)

1. Competent cells preparation (continued)
   • Inoculation of pre-culture SOB from o/n-incubated agar plate colonies.
   • Transfer from pre-culture to growth culture.
2. Colony check
   • All parts successfully transformed
3. Transfer to LB culture medium:1-9N,1-2M,1-14K,1-12M,1-1D,1-17P,1-1G,1-5A,2-16,2-18H,2-16N,1-3A,3-6H

August 4 (Thu)

1. OD measurements throughout the day till required OD (0.3~0.7) was obtained.
2. Completion of competent cells according to protocol.
3. Miniprep of 1-9N,1-2M,1-14K,1-12M,1-1D,1-17P,1-1G,1-5A,2-16,2-18H,2-16N,1-3A,3-6H

August 5 (Fri)

1. Restriction digests of 1-9N,1-2M,1-14K,1-12M,1-1D,1-17P,1-1G,1-5A,2-16,2-18H,2-16N,1-3A,3-6H
2. Gel electrophoresis
3. Ligation
   • 01(K): 1-9N(upstream), 1-12M(downstream), 1-5A(vector)
   • 02(K): 1-1D(upstream), 1-12M(downstream), 1-5A(vector)
   • 03(K): 1-9N(upstream), 3-6H(downstream), 1-5A(vector)
   • 04(K): 1-1D(upstream), 3-6H(downstream), 1-5A(vector)
   • 05(K): 1-1D(upstream), 2-18H(downstream), 1-5A(vector)
   • 06(K): 1-9N(upstream), 2-16L(downstream), 1-5A(vector)
4. Transformation of Registry parts (See Table 2).

Table2

ID	Part Name	Resistance	Description
1-23L	<bbpart>BBa_B0015</bbpart>	A	double terminator

August 6 (Sat)

1. Transfer to LB culture medium:1-23L
2. Transformation of Ligation products
3. Preparation of LB plates (33 Amp, 14 Kan)

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