Gen enes t s to J Jea eans A A Green een S Solution t to - PowerPoint PPT Presentation

Gen enes t s to J Jea eans A A Green een S Solution t to Blue e Den enim UC Berkeley iGEM Bernardo Cervantes Hojae Lee Roy Park Ramya Prathuri Thomas Rich

3,000,000,000 jeans per year 40,000 Steingruber, Indigo and Indigo Colorants (2004) tons of indigo per year Steingruber, Indigo and Indigo Colorants (2004) 66,000,000,000 dollar denim industry World Denim Production and Consumption Report 2012 – DenimsandJeans.com

Indi digo i is synt nthes esized zed from om o oil H 2 SO 4 petroleum HNO 3 ClCH 2 COOH KOH NaOH indigo

Jeans m must b t be dyed with a a soluble f form o of ind ndigo Na 2 S 2 O 4 Air (O 2 )

We w want nt t to d dye e jean ans s sustainab nably 1. Make soluble indigo 2. Dye using our soluble form of indigo

Consulted experts on design principles Key Design Considerations: Jesse Genet Consumers like green • products Most consumers won’t • pay (much) more for green products Dr. Julio Baez Anaerobic conditions can • lower fermentation cost Secretion reduces • process cost ABPDU

Plan ants m mak ake a e a soluble e form o of i indi digo Healthy Leaf p450 GT indole indican (soluble) indoxyl Stressed Leaf Polygonum GLU tinctorium indoxyl indican (soluble) O 2 indigo (insoluble)

The he en enzymatic ic dyein eing meth ethod GT FMO GLU indole indoxyl indican (soluble) O 2 indigo (insoluble) FMO from M. aminisulfidivorans – BBa_K1131000 GT from S. antibioticus – BBa_K1131006 GLU from B. circulans – BBa_K1131002

Mak Making ng indox oxyl yl wi with F FMO FMO indole indoxyl O 2 indigo dye Flask of bio-indigo Best Titer of indigo: 230 mg/L

Char aracter erizat zation on o of F FMO MO Kinet netics cs

Char aracter erizat zation on o of F FMO MO Kinet netics cs

Char aracter erizat zation on o of F FMO MO Kinet netics cs

Char aracter erizat zation on o of F FMO MO Kinet netics cs

Char aracter erizat zation on o of F FMO MO Kinet netics cs K m = 0.87 mM

The he en enzymatic ic dyein eing meth ethod GT FMO GLU indole indoxyl indican (soluble) O 2 indigo (insoluble)

The he indican production p pathway FMO GT indole indoxyl indican (soluble) Paper describing Protein Sequence Protein Database GT sequence data does not exist. activity MATRIAILGAGPSGMAQLRAQS AQEKGAEIPELVCFEKQADWG  QWNYTWRTGLDENGEPVHSS Accession MYRYLWSNGPKECLEFADYTF ID DEHFGKPIASYPPREVLWDYIK GRVEKAGVRKYIRFNTAVRHVE FNEDSQTFTVTVQDHTTDTIY SEEFDYVVCCTGHFSTPYVPEFE GFEKFGGRILHAHDFRDALEFK DKTVLLVGSSYSAEDIGSQCYK YGAKKLISCYRTAPMGYKWPE NWDERPNLVRVDTENAYFAD

The he indican production p pathway FMO GT indole indoxyl indican (soluble) Finding a missing activity: 1. Screening cDNA libraries 2. T esting GTs that act on similar substrates

Sc Screening cDNA NA libraries Indigo producing plant mRNA cDNA Multiple Sequence Alignment B-glucosyl transferases

Char aracter erizi zing ng previou ously u unk nkno nown G n GTs New Glucosyl Transferases are being characterized and submitted to NCBI

Tes esting ng OleD eD: : a pr promiscuous G GT T OleD (Purified Enzyme) OleD No indican Indoxyl (Acceptor) 16 hrs Aerobic reaction UDP-Glucose (Donor)

Two h hyp ypothese ses Hypothesis 1: Aerobic conditions disfavor indican production Hypothesis 2: Native OleD does not recognize indoxyl Active site modifications Thorson et.al, 2010 OleD OleD-ASP

Tes esting ng o our h hypothes eses OleD or OleD ASP (Purified Enzymes) ? Indoxyl (Acceptor) 16 hrs Anaerobic reaction UDP-Glucose (Donor)

Indican i is pr prod oduced ed un under er a anaer aerob obic c condition ons OleD + Indoxyl + UDPG OleD + Indoxyl

Indican i is pr prod oduced ed un under er a anaer aerob obic c condition ons OleD + Indoxyl + UDPG First demonstration of indican production with a recombinant GT! 

B-gluco cosidas dase to dye j e jeans ans GT FMO GLU indole indoxyl indican (soluble) O 2 indigo (insoluble)

B-Glucosidase produces i indigo from i indican GLU KanR ColE1 0 min 10 min 30 min 50mM Indican + GLU lysate = Indigo

Char aracter erizi zing ng B B-glucos cosida dase kinet netics cs K m = 1.78 mM

Can w n we d dye e cloth w with B-gl glucos osidase? 100% Cotton Cloth 100% Cotton Cloth 10 g/L Indican 1 g/L Indican 3.4 g/L B-Glucosidase 3.4 g/L B-Glucosidase Dark jeans Light jeans

Thes ese c e color ors don on’t ’t ru run! H 2 O Acetone 1% SDS 70% EtOH

From t tiny y be bears t rs to t thousa sands o s of j jeans Dip fabric in indican Dip/spray fabric with GLU, dry in air

Engineer neering ng a a cost-effect ective s e system em • Secretion of GLU • Anaerobic production of indican • Overproduction of UDPG • Overproduction of tryptophan

Accom complishmen ents Added 5 well-characterized parts to the registry and a novel protein to NCBI Produced indican for the first time using a recombinant protein Dyed fabric using indican and GLU Developed a novel, green, scalable dyeing process that is both green

Ack ckno nowled edgem emen ents

Ack ckno nowled edgem emen ents! Thanks to the Dueber Lab and EBB for hosting us! Special thanks to: John Dueber T erry Johnson Zach Russ Chris Eiben

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