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

Previo ious iGEM te team ams want nted ed to to u use i e ind ndigo a as a a reporter er Chalmers 2012 KAIST 2012 Cornell 2012 No confirmed indigo No submitted part or No confirmed indigo production in E. coli characterization of the production in yeast indigo production

3,000,000,000 jeans per year Steingruber, Indigo and Indigo Colorants (2004) 40,000 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

Ind ndustri rial indi ndigo i is synt nthesize zed f from o oil O 2 N - phenyl indoxyl benzene aniline indigo glycine

Reduced, s soluble i indigo i is required t to d dye j jeans ns Na 2 S 2 O 4 indigo leuco-indigo O 2 Air (O 2 )

Ind ndustrial i ind ndigo s synt nthesis a and nd d dyeing ng uses ha us hazar ardou ous c chem hemicals H 2 SO 4 HNO 3 ClCH 2 COOH KOH NaOH Na 2 S 2 O 4

Our G r Goals 1) Characterize bio-indigo production HPLC Titer Kinetics 2) Dye jeans using synthetic biology

Plant nts s store s solubilize zed i ind ndigo Healthy Leaf p450 GT indole indoxyl indican indican (soluble) Stressed Leaf GT Polygonum p450 GLU tinctorium indole indoxyl indican (soluble) indican O 2 indigo (insoluble) indigo

Ind ndigo s o synt nthesis and nd d dyeing ng wi with E. . col oli GT FMO GLU indole indoxyl indican (soluble) O 2 indigo (insoluble) FMO from M. aminisulfidivorans – BBa_K1131000 GLU from B. circulans – BBa_K1131002 GT from S. antibioticus – BBa_K1131004 & BBa_K1131005

Char aracter erizi zing ng b bio-indi ndigo p produ oduct ction on GT FMO GLU Indole Indoxyl Indican (soluble) O 2 Indigo dye

Char aracter erizi zing ng b bio-indi ndigo p produ oduct ction on FMO GT GLU Indole Indoxyl Indican (soluble) O 2 Indigo dye

Conf onfirmat ation o n of Indi digo

Conf onfirmat ation o n of Indi digo Indigo Standard

Conf onfirmat ation o n of Indi digo Indigo Standard Bio-Indigo

Indi digo T Titer er

Indi digo T Titer er

Indi digo T Titer er

Indi digo T Titer er

Indi digo T Titer er

Char aracter erizi zing ng o of F FMO MO Kinet netics cs

Char aracter erizi zing ng o of F FMO MO Kinet netics cs

Char aracter erizi zing ng o of F FMO MO Kinet netics cs

Char aracter erizi zing ng o of F FMO MO Kinet netics cs

Char aracter erizi zing ng o of F FMO MO Kinet netics cs K m = 0.87 mM

Findi ding ng a a gl glucos osyl trans ansfer eras ase GT GT FMO GLU Indole Indoxyl Indican (soluble) O 2 Indigo dye GT sequence data does not exist. 

Test sting g homologo gous e s enzym ymes Target substrate: Indoxyl UGT75B2 NP_567471 UGT74D1 Q9LR44 UGT74E2

Sc Screening cDNA NA libraries Indican 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

Br Broad oadeni ening substrat ate s e speci cifici city Active site modifications OleD OleD-ASP A242V S132F P67T Thorson et.al, 2010 Kaempferol Phenol OleD Absorbance Data

In v n vitr tro indi dican can production on Indican Standard MS detection at T R = 10.01min OleD + Indoxyl + UDPG MS detection at T R = 9.93min

Using B ng B-glucosid sidase se to pr produce ce i indigo GT FMO GLU GLU Indole Indoxyl Indican (soluble) O 2 Indigo dye

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

Char aracter erizi zing ng B B-glucos cosida dase kinet netics cs

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 Dyed Cloth Partially Dyed Cloth

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

Impl plica cations o of our b biologica cal d dyeing pr proce cess GT FMO GLU Indole Indoxyl Indican (soluble) O 2 Indigo dye

Human p an prac actices ces • What is its environmental footprint? • Can it be economically competitive? • Talked with bio-production experts at the Advanced Biofuels Process Demonstration Unit

Human p an prac actices ces • Less reliant on petrochemical precursors • Enzyme based, no reduction step necessary • Requires optimization to compete economically with the industrial process

Additions t to o the R Regi gistry Improved Parts BBa_K1131000 M. aminisulfidivorans FMO • BBa_K1131001 M. aminisulfidivorans FMO Mutant • New Parts BBa_K1131004 S. antibioticus OleD • BBa_K1131005 S. antibioticus Oled ASP • BBa_K1131002 B. circulans GLU •

Goin’ ’ for t or the gold gold! Accomplishments:  Improved FMO – characterization and titration data  Preliminary data on indican producing GT – more to come!  Proved fabric can be dyed using indican and GLU  Novel human practices approach

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

Qu Questions? GT FMO GLU Indole Indoxyl Indican (soluble) O 2 Indigo dye