Protocols

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To perform our construct the following procedures

were followed

• 1. Resuspension de biobricks
• 2. Genetic Transformation
• 3. Plasmid Extraction
• 4. Digestion
• 5. Band Purification
• 6. Ligation

Removing long plasmidial

• 1. Inoculate desired strains into 2 ml of Plasmid broth (see Appendix B) plus antibiotic.
• 2. Aerate at 30-37°C until the cultures are fully saturated ( 8 hours to overnight).
• 3. Pour approximately 1.4 ml of culture into a microcentrifuge tube.
• 4. Sediment the cells in a microcentrifuge for 30 seconds.
• 5. Remove the spent culture medium with a water aspirator equipped with a micropipette tip or with a pasteur pipette. Remove as much liquid as possible.
• 6. Resuspend the pellet in 0.1 ml of PEB (see p.502) with gentle vortexing. Make sure that the pellet is well-dispersed. Clumped cells yield decreased DNA.
• 7. Add 0.2 ml of ALM (see p.501) and mix gently by inverting the tube. The solution will clear
• 8. Add 0.15 ml of 3 M sodium acetate (pH 4.8; see p.502). Mix well by inverting and shaking the tube. A flocculent white precipitate will form.
• 9. Sediment the precipitate at 14,000 rpm for 5 minutes. The pellet contains chromosomal DNA and cell debris; the plasmid is in the supernatant.
• 10. Recover 400ul of supernatant into a clean tube containing 0.8 ml of cold 95% ethanol. The insoluble “pellet” tends to float; avoid taking big chunks.
• 11. Chill for 30 minutes to overnight at -20°C. A light-brown DNA precipitate will form. This is a good stopping point; the ethanol precipitate can be left indefinitely at -20°C.
• 12. Sediment the precipitate in a microcentrifuge at 14.000 rpm for 5 minutes.
• 13. Discard the supernatant and rinse the pellet with 1ml of cold 95% ethanol.
• 14. Sediment the pellet in a microcentrifuge at 14.000 rpm for 2 minutes and remove the residual ethanol with a micropipette.
• 15. Dry the pellet in a rotary evaporator for 5 minutes or in air for 30-60 minutes.
• 16. Dissolve the pellet in 0.05ml of TE (see p.502) and store at 4°C. Allow 60 minutes or more for the DNA to go completely into solution.
• 17. Add 0.05ml of 5 M LiCl (see p.502) and mix well.
• 18. Incubate on ice for 5-10 minutes.
• 19. Sediment the precipitate in a microcentrifuge at 14,000 rpm for 5 minutes. The pellet contains RNA and residual cell debris; the plasmid is in the supernatant.
• 20. Recover 100ul of supernatant into a clean tube containing 0.2ml of cold 95% ethanol.
• 21. Chill for 60 minutes to evernight at -20°C. A brown DNA precipitate will form. This is a
good stopping point; the ethanol precipitate can be left indefinitely at -20°C.
• 22. Sediment the precipitate in a microcentrifuge at 14,000 rpm for 5 minutes.
• 23. Discard the supernatant and rinse the pellet with 1ml of cold 95% ethanol
• 24. Sediment the pellet in a microcentrifuge at 14,000 rpm for 2 minutes and remove the residual ethanol with a micro-pipette.
• 25. Dry the pellet in a rotary evaporator for 5 minutes or in air for 30-60 minutes.
• 26. Dissolve the pellet in 0.05ml of TE. The plasmid DNA is now ready for restriction analysis; 5ul per digest is usually sufficient. The DNA is fairly stable at 4°C, but it can be frozen at - 20°C for long-term storage.

Short Plasmid Extraction

• 1. Pelleting 1ml of culture for 5 minutes
• 2. Remove excess. Re-suspend pellet in 250uL suspension (Cell Resuspension Solution)
• 3. Add 250uL of Lysis and invest 4 times .
• 4. Add 10ul Nuclease-Free Water solution. Invest 4 times . Leave for 5 minutes at room temperature
• 5. Add 350ul of Neutralization . Invest 4 times .
• 6. Centrifuge at maximum speed for 10 minutes at room temperature.
• 7. Reverse spin column in the collection tube .
• 8. Discard the clear lysate ( 700ul ) of the spin column
• 9. Centrifuge at maximum speed for 1 minute. Discard it below.
• 10. Stick on 750ul of Wash Solution (ethanol). Centrifuge at maximum speed for 1 minute. Discard supernatant.
• 11.Stick on 250uL of Wash Solution . Centrifuge at maximum speed for 1 minute. discard supernatant
• 12.Transfer to a tube
• 13.Add 80ul of H2OMQ . Centrifuge for 1 minute.

Genetic Transformation Protocol

Add 250 ul of chemocompetente bacteria to an Eppendorf tube Add 2 ul of plasmid ( BioBrick ) Take to the ice for 10 minutes. Take to thermoblock for 50 seconds at 42°C 2 minutes at 0°C Leave it for 10 minutes at room temperature Add 5 ml in a Falcon tube (15 ml ) and 5 ul of antibiotic Insert the material from Eppendorf tube to falcon tube. Incubate 24-48 hours at 37 ° C Genetic transformation (according to IGEM )

• 1. Add 50 ul of chemocompetente to an Eppendorf tube
• 2. Add 2 ul of plasmid (mix with little tap)
• 3. Stand on ice for 20-30 minutes.
• 4. 42°C heat shock for a minute
• 5. Take to the ice for 2 minutes
• 6. Insert 200 ul of LB
• 7. Take it to thermoblock at 37 ° C for 20-30 minutes.
• 8. Add 5 ml of LB to a Falcon tube (15 ml) and lead plate with 5 ul of antibiotic
• 9. Insert the the eppendorf tube material to falcon tube
• 10. Incubate at 37 ° C for 24-48 hrs

P R O T O C O L

DNAPurificationbyCentrifugation

GelSlice andPCR ProductPreparation

A. Dissolving the GelSlice

1. Followingelectrophoresis,exciseDNAband from gelandplace gel slicein a 1.5ml microcentrifuge tube.

2. Add 10µl MembraneBindingSolution per 10mg of gel slice.Vortex and incubate at 50–65°C until gel slice is completely dissolved. B. Processing PCR Amplifications 1. AddanequalvolumeofMembraneBindingSolutiontothePCRamplification. Binding of DNA 1. InsertSVMinicolumn intoCollectionTube. 2. TransferdissolvedgelmixtureorpreparedPCRproducttotheMinicolumn assembly. Incubate at room temperature for 1 minute. 3. Centrifuge at 16,000× g for1 minute.Discard flowthrough and reinsert Minicolumn intoCollection Tube. Washing 4. Add 700µl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube. 5. RepeatStep4with500µlMembraneWashSolution.Centrifugeat16,000×g for 5 minutes. 6. Empty the Collection Tube and recentrifuge the column assembly for 1 minute withthemicrocentrifugelidopen(or off)toallowevaporationofanyresidual ethanol. Elution 7. CarefullytransferMinicolumntoaclean1.5mlmicrocentrifugetube. 8. Add50µlofNuclease-FreeWatertotheMinicolumn.Incubateatroom temperature for1 minute.Centrifuge at 16,000 × g for1 minute. 9. Discard Minicolumn and store DNA at 4°C or –20°C