Team 2 Notes

TODO

1. Find Data about Pbad. How much arabinose is required for expression?
   • Maybe 1 millimolar? See http://2009.igem.org/Team:PKU_Beijing/Project/AND_Gate_1/Inducible_System_Result#Arabinose_Sensor
2. Find Data about Ptet. How much tetrocycline is required for expression?
3. Decide on a buffer. Similar to vacuole? Similar to Cytoplasm?
4. Finish powerpoint.
5. Test cell lysis device?

3/29/10: Protocol planning

Assumed starting materials:

• composite part (CP) w/ genR flanked by terminal repeats, self lysis device, and prepro-transposase under conditional origin of replication (R6K)
• arabinose (how much?)
• "vacuole" buffer (VB)
• pUC-ampR plasmid (how much?)
• tetracycline (how much?)

Steps:

1. Transform a Pir strain with CP, following standard protocol. Pir necessary for plasmid to replicate because it contains a R6K origin of replication. Plate cells on gen, and select colonies. Colonies should be grown in a media containing ___uL arabinose, as the transposase is under a pBad promoter.
2. Create lysis buffer by adding ___mL of VB, ___uL of pUC-ampR plasmid, ____uL of tetracycline (self-lysis device is under pTet promotor), and ___uL of arabinose (transposase is under pBad promoter)
   • Research promoters to find optimal amount of inducer necessary and research transposases to find out how much plasmid is necessary.
3. Add cells to lysis buffer and let incubate for ___ min. At what temperature? Agitate? (Need to find conditions that optimize cell lysis and the transposase reaction)
   • (Note: from 2008 Berkely iGEM: The cultures were then incubated at 37 degrees again for 3.5 hours, and the absorbance at 600nm was measured with a Tecan Xfluor4 Safire2 in a Corning Inc. Costar 3603 plate. The data plotted on a log scale is shown below.)
   • Find self lysis protocol and transposase info to determine these conditions
4. Lysate buffer (at this point it contains VB, cell junk, desired plasmid, CP, chromosomal DNA, transposase, tetracyclin, arabinose) needs to be mini-prepped. Use standard mini-prep protocol.
5. Now we have our desired plasmid and original CP in water. Use this solution to transform cells using the basic protocol. Make sure you DON'T use a strain of Pir cells! We want the CP to fade out.
6. Plate transformed cells on amp/gen plates.
7. Select colonies.
8. Mini-prep and map the plasmids so determine if and where the cassette was inserted.
   • Need to know sequence of DNA that the zinc-fingered transposase binds to and where it inserts transposons

Controls:

1. Testing the R6K origin of replication (To ensure that genR expression in tranformed non-Pir cells is due to transposase activity and NOT successful transformation and replication of the CP)
   • Transform the non-Pir strain with only genR. Use media with arabinose, as in the previous protocol (don't want to introduce another variable).
   • Plate on gen.
   • There should be NO colonies. If there are colonies, the conditional origin of rep is not functioning properly.

Useful links

Zinc finger domains project page for reference

Lysis Device info

Zinc finger paper zf+ comes from L^wt, zf- comes from R^wt, binding sites are shown in Figure 4

Paper on pBad: J Bacteriol 177:4121 (1995) PMID 7608087