Boston UniversityStatus

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Revision as of 19:29, 10 June 2007

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What We've Accomplished

Plasmid Choice

Restriction Enzyme Choice

Primer Design

Week's (Ambitious) Goals

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

Error-Prone PCR: Need to Buy

Ligases: Need to Buy?

Short-Term To-Do List

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

[[Boston_UniversityHeatShockProtocol | Calcium Chloride/Heat Shock Plasmid Transformations] Protocol]

Filter Cojugation Protocol Filter conjugation protocol (put this protocol into a link like for the calcium chloride blah blah blah protocol)

We are using conjugation as a means of transferring a plasmid from transformed E.coli into S.oneidensis. The plasmid contains kanamycin resistance, and the S.oneidensis have gentamicin resistance. Thus, a double selection is used to select for S.oneidensis with plasmid (resistant to both kanamycin and gentamicin). During this double selection, the gentamicin will kill any E.coli and the kanamycin will kill S.oneidensis that lacks plasmid.

Add 0.75mL of magnesium sulfate (MgSO4) to a centrifuge tube filter.

Add 9.3 microliters of the donor cells (E.coli BW20767) and 9.3 microliters of the acceptor cells (S.oneidensis AS-92) to the centrifuge tube filter.

Centrifuge until all of the MgSO4 elutes from the column through the filter.

The filter is now covered with the condensed pellet of cells (thus encouraging conjugation)

Remove the filter from the centrifuge tube filter with sterile tweezers.

Incubate the filter (cell-side up) on an LB agar plate for at least 5 hours at 30 degrees C.

Add 1 mL of LB broth to a tube.

Add the filter (now with grown out cell colonies) into the LB broth.

Vortex the tube to release the cell colonies from the filter surface.

Plate the liquid cell stock onto a double selection(kanamycin and gentamicin) plate.

Question and Answer

Can we get the kanR gene with appropriate sticky ends for BsaI and Tth111I? (DB)

yeah, we can get the DNA template for the kanR gene from the lab. we'll need to design some primers to PCR amplify it out with the BsaI and Tth111I cut sites. after cutting with the restriction enzymes, the kanR will have the appropriate sticky ends. frank suggested that we try ligating the kanR gene to the global regulator gene. (ie. kanR ligated to hlyU) and then stick that into the pJQ200. this way we can select for succesfull hlyU transformations with kanamycin

Ok, good. Do those primers still need to be ordered? And if we follow Frank's suggestion, what will go in place of the gtmR gene?

the hlyU-kanR gene will be inserted into gmtR. the sacB will be left untouched if this method is used.

the other method we can do is to just kill the gmtR gene, then insert hlyU into sacB. we will then add sucrose so any bacteria with a sacB site will die. since hlyU will be interrupting the sacB site, successfull transformations with hlyU will survive the sucrose selection

Also, can someone update the primer design section?

Also also, have thank you letters been sent?

i'll get on both of those by monday's end

Relevant Publications and Links

http://www.shewybase.bu.edu

https://www.atcc.org/common/catalog/numSearch/numResults.cfm?collection=mb-vector&atccNum=77482

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