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

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

Jan 12, 2024 329 likes •791 views

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

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SLIDE 1

UC Berkeley iGEM

Gen enes t s to J Jea eans

A A Green een S Solution t to Blue e Den enim

Bernardo Cervantes Hojae Lee Roy Park Ramya Prathuri Thomas Rich

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SLIDE 2

Previo ious iGEM te team ams want nted ed to to u use i e ind ndigo a as a a reporter er

Cornell 2012 KAIST 2012 Chalmers 2012

No confirmed indigo production in E. coli No submitted part or characterization of the indigo production No confirmed indigo production in yeast

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SLIDE 3

3,000,000,000 66,000,000,000

jeans per year tons of indigo per year dollar denim industry

World Denim Production and Consumption Report 2012 – DenimsandJeans.com Steingruber, Indigo and Indigo Colorants (2004) Steingruber, Indigo and Indigo Colorants (2004)

40,000

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SLIDE 4

Ind ndustri rial indi ndigo i is synt nthesize zed f from o

  • il

benzene indigo aniline N - phenyl glycine indoxyl

O2

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SLIDE 5

Air (O2)

Reduced, s soluble i indigo i is required t to d dye j jeans ns

O2

indigo leuco-indigo

Na2S2O4

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SLIDE 6

Ind ndustrial i ind ndigo s synt nthesis a and nd d dyeing ng us uses ha hazar ardou

  • us c

chem hemicals

KOH NaOH H2SO4 HNO3 ClCH2COOH Na2S2O4

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SLIDE 7

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

HPLC Titer Kinetics

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SLIDE 8

Polygonum tinctorium

indole indoxyl indican p450 GT indole indoxyl indican p450 GT GLU indigo O2 Healthy Leaf Stressed Leaf indican (soluble) indican (soluble) indigo (insoluble)

Plant nts s store s solubilize zed i ind ndigo

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SLIDE 9

indole indoxyl indigo (insoluble) indican (soluble)

FMO GT GLU O2

Ind ndigo s

  • synt

nthesis and nd d dyeing ng wi with E. . col

  • li

FMO from M. aminisulfidivorans – BBa_K1131000 GLU from B. circulans – BBa_K1131002 GT from S. antibioticus – BBa_K1131004 & BBa_K1131005

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SLIDE 10

Indole Indoxyl Indigo dye Indican (soluble)

FMO GT GLU O2

Char aracter erizi zing ng b bio-indi ndigo p produ

  • duct

ction

  • n
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SLIDE 11

Indole Indoxyl Indigo dye Indican (soluble)

GT GLU O2

Char aracter erizi zing ng b bio-indi ndigo p produ

  • duct

ction

  • n

FMO

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SLIDE 12

Conf

  • nfirmat

ation o n of Indi digo

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SLIDE 13

Conf

  • nfirmat

ation o n of Indi digo

Indigo Standard

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SLIDE 14

Conf

  • nfirmat

ation o n of Indi digo

Indigo Standard Bio-Indigo

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SLIDE 15

Indi digo T Titer er

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SLIDE 16

Indi digo T Titer er

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SLIDE 17

Indi digo T Titer er

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SLIDE 18

Indi digo T Titer er

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SLIDE 19

Indi digo T Titer er

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SLIDE 20

Char aracter erizi zing ng o

  • f F

FMO MO Kinet netics cs

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SLIDE 21

Char aracter erizi zing ng o

  • f F

FMO MO Kinet netics cs

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SLIDE 22

Char aracter erizi zing ng o

  • f F

FMO MO Kinet netics cs

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SLIDE 23

Char aracter erizi zing ng o

  • f F

FMO MO Kinet netics cs

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SLIDE 24

Char aracter erizi zing ng o

  • f F

FMO MO Kinet netics cs

Km = 0.87 mM

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SLIDE 25

Indole Indoxyl Indigo dye Indican (soluble)

FMO GT GLU O2

Findi ding ng a a gl glucos

  • syl trans

ansfer eras ase GT sequence data does not exist. 

GT

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SLIDE 26

Q9LR44

Test sting g homologo gous e s enzym ymes

Indoxyl Target substrate:

NP_567471 UGT75B2 UGT74E2 UGT74D1

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SLIDE 27

Sc Screening cDNA NA libraries

mRNA cDNA

B-glucosyl transferases

Multiple Sequence Alignment Indican producing plant

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SLIDE 28

Char aracter erizi zing ng previou

  • usly u

unk nkno nown G n GTs

New Glucosyl Transferases are being characterized and submitted to NCBI

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SLIDE 29

Br Broad

  • adeni

ening substrat ate s e speci cifici city

OleD Kaempferol OleD-ASP Phenol

Active site modifications A242V S132F P67T

Thorson et.al, 2010

OleD Absorbance Data

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SLIDE 30

In v n vitr tro indi dican can production

  • n

OleD + Indoxyl + UDPG MS detection at TR = 9.93min Indican Standard MS detection at TR = 10.01min

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SLIDE 31

Indole Indoxyl Indigo dye Indican (soluble)

FMO GT GLU O2

Using B ng B-glucosid sidase se to pr produce ce i indigo

GLU

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SLIDE 32

B-Glucosidase produces i indigo from i indican

0 min 30 min

50mM Indican + B-Glucosidase = Indigo

10 min

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SLIDE 33

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

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SLIDE 34

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

Km = 1.78 mM

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SLIDE 35

Can w n we d dye e cloth w with B-gl glucos

  • sidase?

100% Cotton Cloth 10 g/L Indican 3.4 g/L B-Glucosidase Dyed Cloth 100% Cotton Cloth 1 g/L Indican 3.4 g/L B-Glucosidase Partially Dyed Cloth

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SLIDE 36

Thes ese c e color

  • rs do

don’t t run! n!

H2O 1% SDS 70% EtOH Acetone

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SLIDE 37

Impl plica cations o

  • f our b

biologica cal d dyeing pr proce cess

Indole Indoxyl Indigo dye Indican (soluble)

FMO GT GLU O2

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SLIDE 38

Human p an prac actices ces

  • Talked with bio-production experts at the Advanced

Biofuels Process Demonstration Unit

  • What is its environmental footprint?
  • Can it be economically competitive?
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SLIDE 39

Human p an prac actices ces

  • Requires optimization

to compete economically with the industrial process

  • Less reliant on

petrochemical precursors

  • Enzyme based, no

reduction step necessary

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SLIDE 40

Additions t to

  • the R

Regi gistry

  • BBa_K1131002 B. circulans GLU
  • BBa_K1131000 M. aminisulfidivorans FMO

New Parts Improved Parts

  • BBa_K1131004 S. antibioticus OleD
  • BBa_K1131005 S. antibioticus Oled ASP
  • BBa_K1131001 M. aminisulfidivorans FMO Mutant
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SLIDE 41

Goin’ ’ for t

  • r the gold

gold!

 Improved FMO – characterization and titration data

Accomplishments:

 Proved fabric can be dyed using indican and GLU  Preliminary data on indican producing GT – more to come!  Novel human practices approach

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SLIDE 42

Ack ckno nowled edgem emen ents

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SLIDE 43

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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SLIDE 44

Qu Questions?

Indole Indoxyl Indigo dye Indican (soluble)

FMO GT GLU O2