From 2008.igem.org

Revision as of 20:22, 29 October 2008

Notes

Here's all our lab work: week by week you can find all the 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.













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.

Up

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],[http://partsregistry.org/Part:BBa_I763020 I763020], [http://partsregistry.org/wiki/index.php?title=Part:BBa_I763005 I763005],[http://partsregistry.org/Part:BBa_C0051 C0051] and [http://partsregistry.org/Part:BBa_C0040 C0040]

• 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)

Up

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

• In the beginning we decided to use light stimulation. The light-sensitive protein taken from the registry was not consistent, and also the Biobrik sent back from the registry cause us many problems. Finally we opted for UV stimulation first for the space selectivity.

• 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.
10.C0040 X\P
Consistent part length.

• Calibration of the fluorescence acquisition system

Up

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

HOLIDAY

Up

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

• Problems with restriction enzymes

• Eluition and Amplification from 2008 Registry Collection of [http://partsregistry.org/Part:BBa_J22106 J22106]

• Bacteria growth curves for the types:

1. TOP10
   
2. DH5A
   
3. XL1BLUE
   

Up

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

Starts the protein construct cloning

• Digestion and Control Gel Run of the previous amplified part (J22106)

1. Ligations: [http://partsregistry.org/Part:BBa_I763020 I763020] + [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
5. Gel extraction of the parts

1. Ligation: [http://partsregistry.org/Part:BBa_B0034 B0034]+ GFP T
2. Trasformation of the ligations in E.coli
3. Inoculation and miniprep preparation
4. Enzymatic digestion and construct gel run: RBS GFP T x\p
5. Gel extraction of the parts

1. Ligation: [http://partsregistry.org/Part:BBa_J22106 J22106] and RBS GFP T
2. Trasformation of the ligations in E.coli
3. Inoculation and miniprep preparation
4. Enzymatic digestion and construct gel run: RBS GFP T x\p
5. Gel extraction of the parts

1. Ligations: [http://partsregistry.org/Part:BBa_S0100 S0100] + [http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_C0040 C0040] + [http://partsregistry.org/Part:BBa_B0015 B0015] and RBS + C0040
2. Trasformation of the ligations in E.coli
3. Inoculation and miniprep preparation
4. Enzymatic digestion and construct gel run:S0100 T x\p, C0040 T x\p, RBS C0034 e\s
5. Gel extraction of the parts

• Fluorescence imaging of the J22106 RBS GFP T construc in different conditions:

1. UV irradiation with an exposure time of 5,10,30 seconds and left grow in the dark
2. UV irradiation with an exposure time of 5,10,30 seconds and left grow in the light

Up

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

1. Ligations: B0034 + C0040 T
2. Trasformation in E.coli
3. Inoculation and miniprep preparation
4. Digestion and gel run of the constructs: RBS C0040 T x\p
5. Gel extraction of the parts
6. Ligations: RBS GFP T + S0100, RBS GFP T + RBS C0040
7. Trasformation in E.coli
8. Inoculation and miniprep preparation
9. Digestion and gel run of: RBS C0040 RBS GFP T x\p, S0100 RBS GFP T x\p
10. Gel extraction

• Final cloning step:

1. Ligations: J23118 + RBS GFP T,J23105 + RBS GFP T, J23100 + RBS GFP T
2. Trasformation in E.coli
3. Inoculation and miniprep preparation
4. Digestion and gel run of: [http://partsregistry.org/Part:BBa_K079031 J23118 RBS GFP T], [http://partsregistry.org/Part:BBa_K079030 J23105 RBS GFP T], [http://partsregistry.org/Part:BBa_K079032 J23100 RBS GFP T]
5. Gel extraction

• Bacteria tracking test on agarose gel

• Fluorescence imaging of the construct J22106 RBS GFP T in different conditions:

1. UV irradiation with an exposure time of 5,10,30 seconds in stationary phase and left grow in the dark
2. UV irradiation with an exposure time of 5,10,30 seconds in exponential phase and left grow in the dark

Up

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

Arrival of the operator library ([http://partsregistry.org/Part:BBa_K079045 Lac], [http://partsregistry.org/Part:BBa_K079046 Tet], [http://partsregistry.org/Part:BBa_K079048 LexA], [http://partsregistry.org/Part:BBa_K079047 Lambda]) 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 [http://partsregistry.org/Part:BBa_K079017 Lac Operator3].
3. Single digestion with XbaI and gel run
4. Gel extraction of the upper band containing [http://partsregistry.org/Part:BBa_K079019 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 [http://partsregistry.org/Part:BBa_K079019 Lac Operator1] e [http://partsregistry.org/Part:BBa_K079018 Lac Operator2].
7. Further single digestion with PstI and gel run.
8. Gel extraction of the upper band containing [http://partsregistry.org/Part:BBa_K079018 Lac Operator2]

This protocol was executed for all of the operator library members, [http://partsregistry.org/Part:BBa_K079046 Tet], [http://partsregistry.org/Part:BBa_K079048 Lex] and [http://partsregistry.org/Part:BBa_K079047 Lambda].

• Fluorescence imaging of the construct J22106 RBS GFP T in different conditions:

1. UV irradiation with an exposure time of 1-5-10 seconds in stationary phase and left grow in the dark
2. UV irradiation with an exposure time of 1-5-10 seconds in exponential phase and left grow in the dark

Up

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

• Execution of protocol design for isolation of single Tet, Lex, Lambda operators from the library.

• Fluorescence imaging of the J22106 RBS GFP T construct in different conditions:

1. UV Irradiation with an exposure time of 1-5-10 seconds. Logaritmic phase of growth with different LB volumes and in presence of light
2. UV irradiation with an exposure time of 1-5-10 seconds. Logaritmic phase of growth with different LB volumes and in the dark
3. UV irradiation with an exposure time of 1-5-10 seconds. Logaritmic phase after different grow times

Up

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

• Assembly of the constructs

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

• Fluorescence imaging of the J22106 RBS GFP T construct in different conditions:

1. After anaerobic growth
2. After aerobic growth

• Bacterial growth in an environment saturated with nitrogen

Up

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

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: [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]

• Fluorescence imaging of the J22106 RBS GFP T construct in different conditions:

1. After growth in an enviroment satured with nitrogen and irradiated with UV for 1,10,15 seconds
2. After growth in standard condition and irradiated with UV for 1,10,15 seconds
3. After growth in standard condition in a batch

Up

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

• Start preparing to [http://partsregistry.org/Part:BBa_K079040 LEXA_2] operator reporter construct:

1. X/P digestion of B0034-J04031-B0010-B0012
2. S/P digestion of [http://partsregistry.org/Part:BBa_K079040 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 in E.coli
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 [http://partsregistry.org/Part:BBa_K079049 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 [http://partsregistry.org/Part:BBa_K079048 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.

• Fluorescence imaging of the J22106 RBS GFP T construct in different conditions:

1. After overnight anaerobic growth in a nitrogen saturated enviroment, an UV irradiation for 1,5,10,15,30 seconds was performed, interposing different water thicknesses between the sample and the UV lamp
2. After overnight anaerobic growth in an nitrogen saturated enviroment,an UV irradiation for 1,5,10,15,20 minutes was performed

Up

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

1. Ligation: J23100 + LexA2 RBS GFP T
2. Trasformation in E.coli
3. Inoculation and miniprep preparation
4. Digestion and gel run
5. Gel extraction of: [http://partsregistry.org/Part:BBa_K079050 J23100-LEXA_2-B0034-J04031-B0010-B0012]

• Fluorescence imaging of the [http://partsregistry.org/Part:BBa_K079050 J23100-LEXA_2-B0034-J04031-B0010-B0012] construct in different conditions:

1. UV irradiation with an exposure time of 10,15,20 minutes. Logaritmic phase of growth and in prsence of light
2. UV irradiation with an exposure time of 10,15,20 minutes. Logaritmic phase of growth and in the dark
3. UV irradiation with an exposure time of 10,15,20 minutes. Logaritmic phase of growth with different LB volumes

Up

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

• Starting from our operator library, we need to extract every single operator to insert these in standard plasmids.

To achieve that we try some methods:

1. Agarose gel 3% for electrophoresis run: no results, the bands are too feeble and yield problem with QIA quick gel extraction kit. The spin columns are optimized for parts ≥ 60bp.
2. Low melting gel 3% and extraction with Phenol Clorophorm: no results, complicated method and high toxicity.

Developed of a new experimental protocol in course of study at our laboratory

• Fluorescence imaging of the [http://partsregistry.org/Part:BBa_K079049 J23118-LEXA_2-B0034-J04031-B0010-B0012] construct in different conditions:

1. UV irradiation with an exposure time of 12 minutes. Logaritmic phase in a LB volume of 1 ml and left grow in the dark

• Sample UV irradiation in different media:

1. Petri dish
2. 15ml tube
3. Agarose gel matrix

Up

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

• Fluorescence imaging of the [http://partsregistry.org/Part:BBa_K079049 J23118-LEXA_2-B0034-J04031-B0010-B0012] construct in different conditions:

1. UV irradiation with an exposure time of 10,20,30 seconds. Logaritmic phase in a LB volume of 1 ml and left grow in the dark
2. UV irradiation with an exposure time of 1,5 seconds. Logaritmic phase in a LB volume of 1 ml and left grow in the dark

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