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From 2013.igem.org

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May 20:

• Organized Gilmer 147 lab space

• Tour of Gilmer lab facilities from Dr. Kay Christopher

• Discussed work schedules. Typically, there will be people in the lab from 9am to 9pm,

working staggered 8-hour shifts.

• Finance: Received $7500 from Medical School, expecting $25,000 each from College

and Engineering (total: $65,000 raised, $9000 left over from last year)

• Reviewed and discussed project proposals.

• List of buffers we need:

May 21:

• Discussed modified CRISPR idea. This is different from the original project proposal.

We would abandon the Cmr-crRNA complexes and use the MS2 coat proteins to export

shRNA sequences via use of a CRISPR array. This array consists of a promoter with

Cas6 (cleaves repeats in the array) and shRNA sequences that are complimentary to

knockdown targets. The double stranded RNA is then cleaved by a eukaryotic cell’s

natural Dicer system.

• If we wanted to use a human cell line (BSL-2), we would have to write it into our PI’s

(Kozminski’s) protocol and get approval from Environmental Health and Safety. Dr.

Christopher says that HeLa cells are easy to grow, but generally difficult to transfect.

However, mouse cell lines are BSL-1 and are easily transfected and there is a lot of

literature available to see if particular lines can express whatever surface receptors (ß-

1, etc.). We will need to talk to Kozminski to obtain BSL-2 approval, however, since

transfecting E. coli with invasin will allow them to be phagocytosed by eukaryotic cells.

• Kozminski says we will need to determine BSL’s and clearly outline our protocols

before contacting EHS. We should do this via him or Dr. Christopher.

• Description of biosafetly levels: http://www.cdc.gov/biosafety/publications/bmbl5/

BMBL5_sect_IV.pdf

• Shaun, Josh, Matt, Greg, Chris L, Jon, Bethany and Liz completed BSL-1 training

List of to Do’s

- PTAO number

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You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.