Team:Osaka/week1

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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
IDPart NameResistanceDescription
1-9N<bbpart>BBa_J22106</bbpart>A rec A (SOS) Promoter
1-2M<bbpart>BBa_K118022</bbpart>ARBS
1-14K<bbpart>BBa_B0040</bbpart>Agreen fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP
1-12M<bbpart>BBa_E0240</bbpart>AGFP generator
1-1D<bbpart>BBa_R0010</bbpart>Apromoter(lacI regulatd)
3-6H<bbpart>BBa_K274100</bbpart>ACrtEBI with rbs
2-18H<bbpart>BBa_K118014</bbpart>Arbs+crtE (geranylgeranyl pyrophosphate synthase)
2-16N<bbpart>BBa_K118006</bbpart>Arbs+crtB (phytoene synthase)
2-18H<bbpart>BBa_K118005</bbpart>Arbs+crtI (phytoene dehydrogenase)
1-17P<bbpart>BBa_I742155</bbpart>AcrtY (lycopene cyclase) with native rbs
1-1G<bbpart>BBa_J04450</bbpart>Aconstruction plasmid containing mRFP coding device
1-3A<bbpart>BBa_J04450</bbpart>Cconstruction plasmid containing mRFP coding device
1-5A<bbpart>BBa_J04450</bbpart>Kconstruction 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
IDPart NameResistanceDescription
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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