Team:Bologna/Notebook

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Revision as of 18:04, 28 October 2008

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


Contents

Appetizer

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Here's all our lab work: week by week you can find all the adopted procedures, links to the registry of standard parts and protocols. The chronological structure of this section, organized as a notebook, mirrors the real development of our project and respects the pure iGEM style.

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.[http://partsregistry.org/Part:BBa_S0100 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

Starts the protein construct cloning

  1. Ligations: [http://partsregistry.org/Part:BBa_I763020 I763020] + [http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_S0100 S0100] + [http://partsregistry.org/Part:BBa_B0015 B0015], TETR + [http://partsregistry.org/Part:BBa_B0015 B0015]
  2. Trasformation of the ligations in E.coli
  3. Inoculation and miniprep preparation
  4. Enzymatic digestion and construct gel run: GFP T x\p, S0100 T x\p, TETR T x\p
  5. Gel extraction of the parts

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

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

  • Protocol design for isolation of single operators from the library.
  1. Single digestion with PstI and gel run. In this way we open the plasmid in 3 points,loosing the Lac Operator1 and 2, and keeping the lac Operator 3 into the plasmid.
  2. Gel extraction of the upper band containing Lac Operator3.
  3. Single digestion with XbaI and gel run
  4. Gel extraction of the upper band containing Lac Operator1.
  5. Single digestion with EcoRI and gel run. In this way we open the plasmid in 2 points,loosing the Lac Operator3, remaining the lac Operator1 and 2 into the plasmid.
  6. Gel extraction of the upper band containing Lac Operator1 e Lac Operator2.
  7. Further single digestion with PstI and gel run.
  8. Gel extraction of the upper band containing Lac Operator2

This protocol was executed for all of the operator library members, Tet, Lex and Lambda.

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

  • Assembly of the constructs
  1. Ligations: Lac2 operator + S0100 RBS GFP T, Lac2 operator + S0100, Lac1 operator + S0100, LexA2 operator + RBS GFP T
  2. Trasformation in E.coli
  3. Inoculation and miniprep preparation
  4. Digestion and gel run
  5. Gel extraction of: LexA2 RBS GFP T x\p, Lac2 S0100 T x\p, Lac2 S0100 RBS GFP T x\p, Lac1 S0100 T x\p

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

  1. Ligation of the previous purified constructs and the promoters J23118, J23100
  2. Trasformation in E.coli
  3. Inoculation and miniprep preparation
  4. Digestion and gel run
  5. Gel extraction of: J23118 S0100 RBS GFP T, J23118 Lac2 S0100 RBS GFP T, J23118 LexA2 RBS GFP T, J23100 LexA2 RBS GFP T, J23118 Lac2 S0100 T, J23118 Lac1 S0100 T

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:
  1. X/P digestion of B0034-J04031-B0010-B0012
  2. S/P digestion of LEXA_2 operator
  3. gel run of B0034-J04031-B0010-B0012 X/P digested and LEXA_2 operator S/P digested
  4. gel extraction of B0034-J04031-B0010-B0012 X/P digested and LEXA_2 operator S/P digested
  5. ligation: B0034-J04031-B0010-B0012 X/P digested + LEXA_2 operator S/P digested
  6. trasformation of LEXA_2-B0034-J04031-B0010-B0012
  7. inoculation of LEXA_2-B0034-J04031-B0010-B0012
  8. miniprep of LEXA_2-B0034-J04031-B0010-B0012
  9. X/P digestion of LEXA_2-B0034-J04031-B0010-B0012
  10. S/P digestion of J23118
  11. gel run of LEXA_2-B0034-J04031-B0010-B0012 X/P digested and J23118 S/P digested
  12. gel extraction of LEXA_2-B0034-J04031-B0010-B0012 X/P digested and J23118 S/P digested
  13. ligation: LEXA_2-B0034-J04031-B0010-B0012 X/P digested + J23118 S/P digested
  14. trasformation of J23118-LEXA_2-B0034-J04031-B0010-B0012
  15. inoculation of J23118-LEXA_2-B0034-J04031-B0010-B0012
  16. miniprep of J23118-LEXA_2-B0034-J04031-B0010-B0012
  17. UV testing of J23118-LEXA_2-B0034-J04031-B0010-B0012
  • since test construct was successfully working, we planned to clone the same construct for the other two LEXA operators to test the repressor- operator binding affinity, in order to choose the one that better suites the implementation of the bistable toggle switch.

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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