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	== '''Primer list''' ==		== '''Primer list''' ==
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-	== '''Plasmid strategy''' ==
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-	http://www.springerlink.com/content/3fj1xxl9p42kx8l5/

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Revision as of 09:59, 19 September 2011

Material and methods

Protocols Plasmid list Primer list

The model of our system consists of three connected modules: the sensor, the band detector and the filter. The sensor detects toxic substance from the cigarette smoke (acetaldehyde or xylene). The band detector produces green fluorescent protein upon a detection of a certain range (band) of toxic substance concentration. After the toxic substance concentration passes a certain threshold the filter turns the system red (red fluorescent protein is expressed).

Protocols

Double digest for cloning

	Variant 1	Variant 2
Plasmid	2 µl EcoRI 2 µl XbaI 5 µl buffer 4 0.5 µl BSA	3 µl PstI 2 µl Spe I 5 µl buffer 2 0.5 µl BSA
Insert	2 µl EcoRI 3 µl Spe I 5 µl buffer 1 0.5 µl BSA	3 µl XbaI 2 µl Pst 1 5 µl buffer 3 0.5 µl BSA

Variant 1

Plasmid

• 2 µl EcoRI
• 2 µl XbaI
• 5 µl buffer 4
• 0.5 µl BSA

Insert

• 2 µl EcoRI
• 3 µl Spe I
• 5 µl buffer 1
• 0.5 µl BSA

Ligation

1. Ligation mix

• 1:5 ratio of plasmid backbone : Insert
• 1 µ 10 x T4 Ligase buffer (vortex)
• 0.5 µ T4 Ligase

adjust to 10 µl with ddH₂O

1. Let the mixture stay for 1h at room temperature
2. Denature the ligase at 65 °C for 5 min

Transformation

1. Thaw competent cells on ice.
2. Add 200 µl cells to 10µl liagtion mix
3. Place the mixture put them on ice for 30 min
4. Heatschock 30 sec at 42 °C
5. Place the mixture again on ice for 2 min
6. Add 4 x SOC medium (800 µl)
7. Let them growth for 60 min at 37°C
8. Spin cells down for 5 min at low speed
9. Remove the supernatant, resuspend the cells
10. Spread 100 &micor;l of the cells onto the plates.

Used parts from registry

• BBa R0040 P_Tet
• BBa R0051 λ_P
• BBa R0010 P_lac
• BBa R0061 P_lux
• BBa J23100 P_const
• BBa C0061 LuxI_LVA
• BBa C0040 TetR_LVA
• BBa C0051 CI
• BBa C0062 LuxR
• BBa B0015 Double Terminator

Plasmid list

Primer list

Synthesized parts

• LacI_M1
• AlcR (codon optimized)

Characterization of LacI_M1