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	== Question and Answer ==		== Question and Answer ==
		+	According to ATCC, plasmid PJQ200 "replicates only in enterobacteria, and so functions as a suicide vector in other Gram negative hosts." Since shewy is not an enterobacterium, how will PJQ200 help us get that gene into shewy and its future generations?
	== Relevant Publications and Links==		== Relevant Publications and Links==

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Revision as of 13:32, 8 June 2007

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Contents 1 What We've Accomplished 2 Week's (Ambitious) Goals 3 Materials We Need 4 Short-Term To-Do List 5 Protocols 6 Question and Answer 7 Relevant Publications and Links

What We've Accomplished

Plasmid Choice

Restriction Enzyme Choice

Primer Design

Week's (Ambitious) Goals

Wednesday 5/30

1. Get all protocols
2. Identify materials/prepare order
3. Design Primers
4. Learn about budget/POs

Thursday 5/31

1. Do primer order
2. Start conjugation practice
3. Confirm restriction enzymes, ligases
4. Order confirmed/needed materials
5. Team Revew Meeting
6. Draft Thank-You Letters for our Sponsors

Friday 6/1

1. Evaluate/continue conjugation, practice electroporation for E. coli
2. Revise proposal to include possibility of screening with alginate beads and fluorocytometer
3. Meeting with Tim: Budgets/protocols, Pfizer/fundraising, iGEM registration, beads

Week of 6/4:

1. Evaluate the transformation that was done on Friday.

2. Confirm the correct plasmid (pJQ200)

3. Find appropriate restriction enzymes that cut by BLASTing all the plasmid's restriction enzyme sites onto the global transcription factors.

4. Re-design primers for the global transcription factors based on the restriction enzymes we have selected.

5. Order primers.

6. Practice regular (non-error prone) PCR with the primers to check that they work.

7. Incorporate the global transcription factors into the plasmid and transform this plasmid into the E.coli.

8. Conjugate this plasmid into Shewy.

Materials We Need

Primers: BOUGHT!

Error-Prone PCR: Need to Buy

Plasmids: BOUGHT!

Restriction Enzymes: Need to Buy?

Ligases: Need to Buy?

Short-Term To-Do List

Lab Orientation: COMPLETED!

• Lab Orientation Checklist

Lab Safety Training: COMPLETED!

Design of Primers: COMPLETED!

Ordering of Primers: COMPLETED!

Gathering of Protocols: Not Completed (Chris, please send me the protocols when they are gathered)

Ordering of Error-Prone PCR Materials: Not completed

Thank-You Letters sent to Pfizer: Not Completed

Thank-You Letters sent to BU ppl: Not completed

Protocols

"Calcium Chloride/Heat Shock Plasmid Transformations"

Question and Answer

According to ATCC, plasmid PJQ200 "replicates only in enterobacteria, and so functions as a suicide vector in other Gram negative hosts." Since shewy is not an enterobacterium, how will PJQ200 help us get that gene into shewy and its future generations?

Relevant Publications and Links

http://www.shewybase.bu.edu

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