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Contents 1 Lac Operon Control 2 Ara Operon Control (pbad promoter) 3 RBS Selection 4 Chitin 4.1 Chitin Synthesis 4.2 Chitin Staining (Calcofluor) 5 Apoptosis 5.1 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

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

RBS Selection

http://partsregistry.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Community_Collection

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 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

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/

Apoptosis

registry: http://partsregistry.org/Cell_death

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

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

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