Team:Northwestern/References

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1 Lac Operon Control
2 Ara Operon Control (pbad promoter)
3 PhoB Promoter
4 Chitin
- 4.1 Chitin Synthesis
- 4.2 Chitin Staining (Calcofluor)
5 Apoptosis
- 5.1 Control
- 5.2 Live/Dead Staining aka Viability/Cytotoxicity Assay
6 Keio Collection - KnockOuts
- 6.1 ChiA(Chitinase) Knockout (may not be necessary)
- 6.2 ydgg/tqsA (AI2-Transporter) Knockout
7 BIF (Biological Imaging Facility)

Lac Operon Control

http://openwetware.org/wiki/IPTG

Ara Operon Control (pbad promoter)

http://partsregistry.org/wiki/index.php?title=Part:BBa_R0080

http://partsregistry.org/wiki/index.php/Part:BBa_I0500

arabinose information: http://openwetware.org/wiki/Arabinose

pbad info: http://openwetware.org/wiki/Titratable_control_of_pBAD_and_lac_promoters_in_individual_E._coli_cells#pBAD_promotersOpenWetWare

pbad family (only wildtype available) - http://partsregistry.org/PBAD_Promoter_Family

PhoB Promoter

http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00053

Chitin

Adding Glucosamine to growth medium boosts chitin production in Yeast (S.C.)- http://ec.asm.org/cgi/reprint/2/5/886

Chitin synthesis Pathway (image) - [http://www.mekarn.org/msc2005-07/thesis07/latslrimage002.jpg] (Figure.1. Biosynthesis of chitin in insects. The pathway starts with trehalose, the main hemolymph sugar in most insects, and ends with the chitin polymer. The diagrammatic representation is based on previously published pathways (Kramer and Koga, 1986; Cohen, 2001).)

Chitin Precursor (UDP-N-Acetyl-Glucosamine) is used for Endotoxin (Lipid A) production - http://www.jbc.org/content/268/26/19858.long - possible safety issue - "When bacterial cells arelysed by the immune system, fragments of membrane containing lipid A are released into the circulation, causing fever, diarrhea, and possible fatal endotoxic shock (also called septic shock)." - thus spake wikipedia

Chitin Uses: http://www.gmp-chitosan.com/en/products-services/chitin.html

Chitin Synthesis

chitin synthase gene (chs3): http://www.ncbi.nlm.nih.gov/gene/852311

CHS3 does polymerization (Chitin-UDP acetyl-glucosaminyl transferase is just another name for CHS3) - http://www.ebi.ac.uk/interpro/IEntry?ac=IPR004835, http://www.uniprot.org/uniprot/P29465

http://tools.neb.com/NEBcutter2/cutshow.php?name=cb3c5ddc-

Chitin Staining (Calcofluor)

Does not stain E Coli - http://www.sigmaaldrich.com/etc/medialib/docs/Fluka/Datasheet/18909dat.pdf

Stains Biofilm Polysaccharides? - http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6V73-3YKKH2J-4S-1&_cdi=5831&_user=965532&_pii=0043135494003399&_orig=search&_coverDate=08%2F31%2F1995&_sk=999709991&view=c&wchp=dGLbVzz-zSkzS&md5=fedcb287ed9c9dafca2dbb4f2bc053a4&ie=/sdarticle.pdf

Inhibits Synthase - http://www.ncbi.nlm.nih.gov/pmc/articles/instance/40653/

Data sheet: https://docs.google.com/viewer?url=http://www.sigmaaldrich.com/etc/medialib/docs/Fluka/Datasheet/18909dat.Par.0001.File.tmp/18909dat.pdf&pli=1

Excitation/Emission: http://www.olympusmicro.com/primer/techniques/fluorescence/fluorotable2.html

Apoptosis

registry: http://partsregistry.org/Cell_death

Control

Adjust genetic order
RBS of varying strengths
OmpR Binding Site (to detect extracellular glucose)
- http://partsregistry.org/wiki/index.php?title=Part:BBa_I761001
- http://partsregistry.org/wiki/index.php/Part:BBa_I761011
PhoB

Live/Dead Staining aka Viability/Cytotoxicity Assay

DMAO(Live), EtD-III(Dead)

http://www.promokine.info/products/cell-analysis/cell-staining-reagents-related-products/
http://www.biotium.com/product/product_info/Protocol/30027.pdf

Keio Collection - KnockOuts

Website: http://ecoli.naist.jp/gb6/Resources/deletion/deletion.html

Ordering: http://www.shigen.nig.ac.jp/ecoli/strain/top/top.jsp

Method: http://www.pnas.org/content/97/12/6640.full.pdf+html

Paper: http://www.nature.com/msb/journal/v2/n1/full/msb4100050.html

Primer Extensions: http://www.biomedsearch.com/attachments/00/16/73/85/16738554/msb4100050-s4.xls

ChiA(Chitinase) Knockout (may not be necessary)

summary of e coli knockouts: http://openwetware.org/wiki/Escherichia_coli/Knockouts

chitinase gene (chiA): http://www.ncbi.nlm.nih.gov/gene/947837

chiA-knockout strain e coli: http://ecoli.naist.jp/GB6/info.jsp?id=JW3300

ydgg/tqsA (AI2-Transporter) Knockout

KO -> ~7000x biofilm thickness, ~500x biomass - http://jb.asm.org/cgi/content/short/188/2/587

http://ecoli.aist-nara.ac.jp/GB5/info.jsp?id=JW1593

more info: http://www.open-access-biology.com/biofilms/biofilmsch7.pdf

BIF (Biological Imaging Facility)

List of Equipment: http://www.northwestern.edu/bioimaging/equip.html
Methods: http://www.northwestern.edu/bioimaging/methods.html