Team 2 Notes

To do

ASAP

• Figure out how much plasmid to add to the lysate (look at protocols)
• Degredation Assay (a day long transformation procedure)
• Figure out what buffer to use (research online)

Once the composite parts are complete

• Transform genR-selflysis-transposase composite part into pir+ strain
• Arabinose assay: test when we should add arabinose and how much we should add to optimize transposase expression (time: an hour before, when we add anhydrotetracycline)-> this will require a day long transformation procedure.
• The actual assay

Degredation Assay

• get transformed cells w/ self-lysis device (how much?)
• need pUC-ampR plasmid
• need anhydrotetracycline and figure out concentration necessary
• Prepare/get the buffer. NEB? PBS+MgCl2?
• Mix it all up
• remove samples at different intervals (5min, 10min, 15, 20, 30, 45, 60)
• Zymo/truncated Mini Prep?
• Transform
• Plate

Next time: Count colonies

Materials: -ampR plasmid -cells w/ self-lysis device? Or will we have to transform them ourselves?

4/7/10: Presentation Feedback

• Use MC1061(pir). This strain doesn't degrade arabinose due to mutations in some of its metabolic pathways.
• Variable: Time at which we add arbinose. Could try adding arabinose at the same time as anhydratetracycline (aTc) or an hour before aTc.
• Could take real cells and generate buffer by grinding them up.
• Don't use PBS since it doesn't have Mg.
• Self-lysis won't take ~3.5 hours. It will probably take a few minutes. NOTE: incubating too long might cause DNA in the buffer to be degraded by nucleases.
• IMPORTANT: determine rate of DNA degradation in the lysate. Could transform cells with lysate samples taken at different times and count colonies.
• Zymo might be fine instead of miniprepping lysate.
• MC1061, DH10B, TG1, all choices for (pir -)
• DPN1 fingerprinting could be used for mapping. Will create ~10-20 cuts in the average plasmid. Do wild-type digest side by side with integrand, compare bands.
• How much plasmid will we add to the lysate?

Completed 3/31/10 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?
   • <50 ng/ml of anhydrotetracycline
   • Check http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040
3. Decide on a buffer. Similar to vacuole? Similar to Cytoplasm?
   • PBS?
   • transposition buffer: 100 mM Tris pH 7.5, 150 mM MgCl2, 100 mM KCl, 10 mM DTT
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

transposition reaction buffer mentioned

[Paper on cytoplasm mimicry]