Team:Bologna/Notebook

 body {background-color:#000000} strong {color: #c09a6d;}

=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: R0082, R0083, 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: E0240, pSB3K3_P1010in DB3.1 and the Practice Promoter Set (J23150, J23151, J23102) to test and set up the new Biobrick Standard Measurement Protocol


 * Transformation and Amplification from our Lab Stock of S0100,I763020, I763005,C0051 and 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.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 J22106


 * Bacteria growth curves for the following strains:
 * 1) TOP10
 * 2) DH5ALFA
 * 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: I763020 + 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: 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: 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: S0100 + B0015, C0040 + 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 construct in different conditions:
 * 1) UV irradiation with an exposure time of 5,10,30 seconds and grow in the dark
 * 2) UV irradiation with an exposure time of 5,10,30 seconds and grow in presence of 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: J23118 RBS GFP T, J23105 RBS GFP T, 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. Stationary phase of growth and in the dark
 * 2) UV irradiation with an exposure time of 5,10,30 seconds. Logaritmic phase of growth and in the dark

Up

= Week 8: from 09/08/08 to 09/14/08 = Arrival of the operator library (Lac, Tet, LexA, 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 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.


 * Fluorescence imaging of the construct J22106 RBS GFP T in different conditions:
 * 1) UV irradiation with an exposure time of 1,5,10 seconds. Stationary phase of growth and in the dark
 * 2) UV irradiation with an exposure time of 1,5,10 seconds. Logaritmic phase of growth and 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: J23118 S0100 RBS GFP T, J23118 Lac2 S0100 RBS GFP T, J23118 Lac2 S0100 T, J23118 Lac1 S0100 T


 * Fluorescence imaging of the J22106 RBS GFP T construct in different conditions:
 * 1) After growth in nitrogen saturated enviroment an UV irradiation for 1,10,15 seconds
 * 2) After growth in standard condition, an UV irradiation 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 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 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 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.


 * 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 a 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: J23100-LEXA_2-B0034-J04031-B0010-B0012


 * Fluorescence imaging of the 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 persence 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 J23118-LEXA_2-B0034-J04031-B0010-B0012 construct in different conditions:
 * 1) UV irradiation with an exposure time of 12 minutes. Logaritmic phase of growth in a LB volume of 1 ml and 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 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 of growth in a LB volume of 1 ml and in the dark
 * 2) UV irradiation with an exposure time of 1,5 seconds. Logaritmic phase of growth in a LB volume of 1 ml and in the dark

Working on our wiki!!!

Up