Team:Bologna/Notebook

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HOME PROJECT TEAM SOFTWARE MODELING WET LAB SUBMITTED PARTS BIOSAFETY AND PROTOCOLS


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Contents

Protocols summary

For more information, see Biosafety page
Protocol Biosafety level
Plates preparation
1
Amplification
1
Transformation
1
Inoculation
1
Miniprep
1
Digestion reaction
1
Gel preparation
2
Electrophoretic run
2
Gel extraction
2
Ligation reaction
1
Chemiocompetent cells
1
Mediums and buffers
1
Antibiotics stocks preparation
1
IPTG stocks preparation
1
Fluorescence test
1
M9 supplemented media
1


Up

Week 1: from 07/21/08 to 07/27/08

General Preparations

  1. Preparation of chemiocompetent cells from E. Coli DH5α, Top10 and DB 3.1
  2. Preparation of antibiotic stocks for Ampicillin and Kanamicin
  3. Preparation of LB medium and LB plates for cloning.

Week 2: from 07/28/08 to 08/03/08

  • Eluition and Amplification from 2008 Registry Collection: [http://partsregistry.org/Part:BBa_R0082 R0082,] [http://partsregistry.org/Part:BBa_R0083, R0083], [http://partsregistry.org/wiki/index.php/Part:BBa_M30109 M30109] in TOP10 strain to build and characterize the Light response system to be our spatial selective trigger.
  • Eluition and Amplification from 2008 Registry Collection: [http://partsregistry.org/Part:BBa_E0240 E0240], [http://partsregistry.org/Part:BBa_P1010 pSB3K3_P1010]in DB3.1 and the Practice Promoter Set ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23103/ J23150, J23151, J23102]) to test and set up the new [http://partsregistry.org/Measurement Biobrick Standard Measurement Protocol]
  • Transformation and Amplification from our Lab Stock of [http://partsregistry.org/Part:BBa_S0100 S0100], BBa_I763020, [http://partsregistry.org/wiki/index.php?title=Part:BBa_I763005 I763005] and [http://partsregistry.org/Part:BBa_C0051 C0051]
  • Growth Curves of Dh5 Alpha, Top10 and XL1 Blue with Low Medium and High Copy Numbers to assay and define the different kinetics (Further Detail)

Week 3: from 08/04/08 to 08/10/08

08/04/08

  • Digestion and Control Gel Run of the previous amplified constructs :

1. S0100 E/S
Consistent Part Length
2. PLAC-CI X/P
Consistent Part Length
3. R0083 S/P
Single Vector Band as Expexted. Is Hard to verify the Part length correctness given the small size
4. R0082 S/P
Single Vector Band as Expexted. Is Hard to verify the Part length correctness given the small size
5. C0051 X/P
Consistent Part Length.
7. M30105 E/S
The Part appears not consistent. The Gel has unexpected multiple bands.
8. RBS GFP TAG X/P
Consistent Part Length
9.Pλ GFP X/P
Consistent Part Length.

  • Ligation of R0082 and R0083 with E0240 to obtain a Reporter for the Light Driven Trigger.

Week 4: from 08/11/08 to 08/17/08

HOLIDAY

Week 5: from 08/18/08 to 08/24/08

Starting development of protein construct

  1. Ligation of I763020 and B0015, of S0100 and B0015, of TETR and B0015
  2. Trasformation of the ligations in E.coli
  3. Inoculation and preparation of miniprep
  4. Digestion and control gel run of the constructs: GFP T x\p, S0100 T x\p, TETR T x\p
  5. Gel extraction of the part

Week 6: from 08/25/08 to 08/31/08

  1. Ligation of B0034 and TetR T , B0034 and GFP T
  2. Trasformation in E.coli
  3. Inoculation and preparation of Miniprep
  4. Digestion and control gel run of the constructs: RBS TETR T x\p, RBS GFP T x\p
  5. Gel extraction of the parts
  6. Ligation of RBS GFP T and S0100, RBS GFP T and RBS TetR
  7. Trasformation in E.coli
  8. Inoculation and preparation of Miniprep
  9. Digestion and control gel run of the parts: RBS TETR RBS GFP T x\p, S0100 RBS GFP T x\p
  10. Gel extraction
  • Termination of the constructs:
  1. Ligation of promotor J23118 and RBS GFP T, promotor J23105 and RBS GFP T, promotor J23100 and RBS GFP T
  2. Trasformation in E.coli
  3. Inoculation and preparation of Miniprep
  4. Digestion and control gel run of the constructs:J23118 RBS GFP T, J23105 RBS GFP T, J23100 RBS GFP T
  5. Gel extraction

Week 7: from 09/01/08 to 09/07/08

Arrival of the operator library (link) from GeneArt

  • Implementation of the protocol to separate each Lac Operator from the library
  1. Single digestion with Pst and control gel run (In this way we open the plasmid in 3 points,leaving free the Lac Operator1 and 2, remaining the plasmid with the lac Operator 3)
  2. Gel extraction of the upper band containing Lac Operator3 inside the plasmid
  3. Single digestion with Xba and control gel run
  4. Gel extraction of the upper band containing Lac Operator1 inside the plasmid
  5. Single digestion with EcoRI and control gel run (In this way we open the plasmid in 2 points,leaving free the Lac Operator3, remaining the plasmid with the lac Operator1 and 2)
  6. Gel extraction of the upper band containing Lac Operator1 e Lac Operator2 inside the plasmid
  7. Further single digestion with Pst and control gel run
  8. Gel extraction of the upper band containing Lac Operator2

This protocol was executed for both Tet, Lex, Lambda operators

Week 8: from 09/08/08 to 09/14/08

Week 9: from 09/15/08 to 09/21/08

Week 10: from 09/22/08 to 09/28/08

Week 11: from 09/29/08 to 10/05/08

Start preparing to LEXA_2 operator reporter construct:

  • first day
  1. digestion X/P of B0034-J04031-B0010-B0012
  2. digestion S/P of LEXA_2 operator
  3. Control Gel Run of B0034-J04031-B0010-B0012 digested X/P and LEXA_2 operator digested S/P
  4. Gel extraction of B0034-J04031-B0010-B0012 digested X/P and LEXA_2 operator digested S/P
  5. ligation of B0034-J04031-B0010-B0012 digested X/P with LEXA_2 operator digested S/P
  6. trasformation of LEXA_2-B0034-J04031-B0010-B0012
  • second day
  1. inoculation of LEXA_2-B0034-J04031-B0010-B0012
  2. miniprep of LEXA_2-B0034-J04031-B0010-B0012
  • third day
  1. digestion X/P of LEXA_2-B0034-J04031-B0010-B0012
  2. digestion S/P of J23118
  3. control gel run of LEXA_2-B0034-J04031-B0010-B0012 digested X/P and J23118 digested S/P
  4. gel extraction of LEXA_2-B0034-J04031-B0010-B0012 digested X/P and J23118 digested S/P
  5. ligation of LEXA_2-B0034-J04031-B0010-B0012 digested X/P with J23118 digested S/P
  6. trasformation of J23118-LEXA_2-B0034-J04031-B0010-B0012
  • fourth day
  1. inoculation of J23118-LEXA_2-B0034-J04031-B0010-B0012
  2. miniprep of J23118-LEXA_2-B0034-J04031-B0010-B0012
  • since construct test was successful, we have proceeded with the preparation of the same construct for the other two LEXA operator

Week 12: from 10/06/08 to 10/12/08

Week 13: from 10/13/08 to 10/19/08

Week 14: from 10/20/08 to 10/26/08

Week 15: from 10/27/08 to 10/29/08


















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