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Team:Bologna/Notebook
From 2008.igem.org
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= Week 12: from 10/06/08 to 10/12/08 = | = Week 12: from 10/06/08 to 10/12/08 = | ||
| - | + | # Ligation of J23100 and LexA2 RBS GFP T | |
| - | + | # Trasformation in E.coli | |
| - | + | # Inoculation and miniprep preparation | |
| - | + | # Digestion and gel run | |
| - | + | # Gel extraction of: J23100 LexA2 RBS GFP T | |
| - | + | ||
= Week 13: from 10/13/08 to 10/19/08 = | = Week 13: from 10/13/08 to 10/19/08 = | ||
Revision as of 19:07, 28 October 2008
| HOME | PROJECT | TEAM | SOFTWARE | MODELING | WET LAB | LAB-BOOK | SUBMITTED PARTS | BIOSAFETY AND PROTOCOLS |
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Appetizer
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
- Preparation of chemiocompetent cells from E. Coli DH5α, Top10 and DB 3.1
- Preparation of antibiotic stocks for Ampicillin and Kanamicin
- 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
- 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]
- Trasformation of the ligations in E.coli
- Inoculation and miniprep preparation
- Enzymatic digestion and construct gel run: GFP T x\p, S0100 T x\p, TETR T x\p
- Gel extraction of the parts
Week 6: from 08/25/08 to 08/31/08
- Ligations: B0034 + TetR T , B0034 + GFP T
- Trasformation in E.coli
- Inoculation and miniprep preparation
- Digestion and gel run of the constructs: RBS TETR T x\p, RBS GFP T x\p
- Gel extraction of the parts
- Ligations: RBS GFP T + S0100, RBS GFP T + RBS TetR
- Trasformation in E.coli
- Inoculation and miniprep preparation
- Digestion and gel run of: RBS TETR RBS GFP T x\p, S0100 RBS GFP T x\p
- Gel extraction
- Final cloning step:
- Ligations: promotor J23118 + RBS GFP T, promotor J23105 + RBS GFP T, promotor J23100 + RBS GFP T
- Trasformation in E.coli
- Inoculation and miniprep preparation
- Digestion and gel run of: J23118 RBS GFP T, J23105 RBS GFP T, J23100 RBS GFP T
- 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.
- 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.
- Gel extraction of the upper band containing Lac Operator3.
- Single digestion with XbaI and gel run
- Gel extraction of the upper band containing Lac Operator1.
- 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.
- Gel extraction of the upper band containing Lac Operator1 e Lac Operator2.
- Further single digestion with PstI and gel run.
- 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
- Ligations: Lac2 operator + S0100 RBS GFP T, Lac2 operator + S0100, Lac1 operator + S0100, LexA2 operator + RBS GFP T
- Trasformation in E.coli
- Inoculation and miniprep preparation
- Digestion and gel run
- 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
- Ligation of the previous purified constructs and the promoters J23118, J23100
- Trasformation in E.coli
- Inoculation and miniprep preparation
- Digestion and gel run
- Gel extraction of: [http://partsregistry.org/Part:BBa_K079026 J23118 S0100 RBS GFP T], [http://partsregistry.org/Part:BBa_K079020 J23118 Lac2 S0100 RBS GFP T], [http://partsregistry.org/Part:BBa_K079023 J23118 Lac2 S0100 T], [http://partsregistry.org/Part:BBa_K079023 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:
- X/P digestion of B0034-J04031-B0010-B0012
- S/P digestion of LEXA_2 operator
- gel run of B0034-J04031-B0010-B0012 X/P digested and LEXA_2 operator S/P digested
- gel extraction of B0034-J04031-B0010-B0012 X/P digested and LEXA_2 operator S/P digested
- ligation: B0034-J04031-B0010-B0012 X/P digested + LEXA_2 operator S/P digested
- trasformation in E.coli
- inoculation of LEXA_2-B0034-J04031-B0010-B0012
- miniprep of LEXA_2-B0034-J04031-B0010-B0012
- X/P digestion of LEXA_2-B0034-J04031-B0010-B0012
- S/P digestion of J23118
- gel run of LEXA_2-B0034-J04031-B0010-B0012 X/P digested and J23118 S/P digested
- gel extraction of LEXA_2-B0034-J04031-B0010-B0012 X/P digested and J23118 S/P digested
- ligation: LEXA_2-B0034-J04031-B0010-B0012 X/P digested + J23118 S/P digested
- trasformation of J23118-LEXA_2-B0034-J04031-B0010-B0012
- inoculation of J23118-LEXA_2-B0034-J04031-B0010-B0012
- miniprep of [http://partsregistry.org/Part:BBa_K079049 J23118-LEXA_2-B0034-J04031-B0010-B0012]
- 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
- Ligation of J23100 and LexA2 RBS GFP T
- Trasformation in E.coli
- Inoculation and miniprep preparation
- Digestion and gel run
- Gel extraction of: J23100 LexA2 RBS GFP T
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
