Ellen Cliff, Conor Horgan, Richard Kong, Henry Orton, Janelle San - - PowerPoint PPT Presentation

Ellen Cliff, Conor Horgan, Richard Kong, Henry Orton, Janelle San Juan, Victor Wang, Laura Wey, Matthew Witney Colin Jackson (Research School of Chemistry), Spencer Whitney (Research School of Biology)

Engineering

Mathematics

Chemistry Biology

WHO ARE WE?

Early engineers Math humor Early chemists describe the first dirt molecule Stimulus response! Stimulus response! Don’t you ever think?

Engineering

Mathematics

Chemistry Biology

WHO ARE WE?

Dr Colin Jackson (ANU Research School of Chemistry) A/Prof Spencer Whitney (ANU Research School of Biology)

WHAT IS PHOTOGENEIC ABOUT?

• ptogenetics: the control of cellular dynamics using light

International Year of Light

LIGHT-INDUCIBLE CRY2/CIB1 SYSTEM

CRY2

Leaf cell

Blue light Dark

CRY2

Active transcription Inactive

FAD

CIB1 CIB1

LIGHT-INDUCIBLE CRY2/CIB1 SYSTEM

• Fundamental strategy: fuse two inactive halves of a

target protein to CRY2 and CIB1 that form an active protein upon blue light induced co-association.

Yeast, zebra fish, leaf cell

Blue light Dark

EP active Effector protein (EP) inactive CRY2 CIB1 CRY2

FAD

CIB1 EPN EPC

APPLYING CRY2/CIB1 in E. coli

• Ultimate objective – light inducible induction of protein

expression for toxic metabolite biosynthesis.

Gene 1 A B C D Desired product Product - Gene - Gene 2 Gene 3 Gene 4 Enzyme1 Enzyme2 Enzyme3 Enzyme4

Cell death

Rate limiting

APPLYING CRY2/CIB1 in E. coli

• Ultimate objective – light inducible induction of protein

expression for toxic metabolite biosynthesis.

nadB L-aspartate α-iminoaspartate quinolate

NAD

nadA NADB NADA [NAD] Cell fitness



APPLYING CRY2/CIB1 in E. coli

• Ultimate objective – light inducible induction of protein

expression for toxic metabolite biosynthesis.

nadB L-aspartate

α-iminoaspartate

quinolate

NAD

NADB

• Proof Of Concept Gaol – high yield NAD biosynthesis

(non-toxic)

nadA NADA

APPLYING CRY2/CIB1 in E. coli

• Ultimate objective – light inducible induction of protein

expression for toxic metabolite biosynthesis.

nadB L-aspartate

NAD

nadA NADB NADA

• Proof Of Concept Gaol – high yield NAD biosynthesis

APPLYING CRY2/CIB1 in E. coli

• Ultimate objective – light inducible induction of protein

expression for toxic metabolite biosynthesis.

• Proof of Concept goal – high yield NAD biosynthesis in E. coli.

CRY2 CIB1 CREN CREC nadB

loxP loxP

P

nadA

L-aspartate α-iminoaspartate

NADB

Blue light Dark

CRY2

FAD

CIB1

Gene 1

loxP

P

nadA nadB

loxP

quinolate NAD+

NADA

Cre-recombinase active

EXPERIMENTAL AIMS

AIM 1. Can we detect CRY2-CIB1 function in E. coli? AIM 2. Can we adapt CRY2-CIB1 to trigger gene recombination? AIM 3. Can we exploit CRY2-CIB1 triggered CRE- recombination for high yield NAD biosynthesis in E. coli?

EXPERIMENTAL AIMS

AIM 1. Can we detect CRY2-CIB1 function in E. coli?

CRY2 CIB1

Blue light Dark

CRY2

FAD

CIB1

FRET

YFP CFP

EXPERIMENTAL AIMS

AIM 1. Can we detect CRY2-CIB1 function in E. coli?

AIM 2. Can we adapt CRY2-CIB1 to trigger gene recombination?

CRY2 CIB1 CREN CREC

gene1

loxP loxP

P

gene2

Enz1

Blue light Dark

CRY2

FAD

CIB1

Gene 1

loxP

P

2 2

loxP

Enz2

EXPERIMENTAL AIMS

AIM 1. Can we detect CRY2-CIB1 function in E. coli?

AIM 2. Can we adapt CRY2-CIB1 to trigger gene recombination? AIM 3. Can we exploit CRY2-CIB1 triggered CRE- recombination for high yield NAD biosynthesis in E. coli?

CRY2 CIB1 CREN CREC nadB

loxP loxP

P

nadA NADB

L-aspartate α-iminoaspartate

Blue light Dark

CRY2

FAD

CIB1

Gene 1

loxP

P

nadA nadB

loxP

NADA

quinolate NAD+

METHODS

Modular gene expression

CRY2 [1] Kennedy et al., (2010) Nature Methods 7, 249–252 [2] Meng et al., (2013) Plant Cell 25, 4405-20

Genes coding the A. thaliana N-domain of CRY2 and CIB1 that are required for blue light interaction [1,2] were synthesized by GenScript

CRY2

FAD

1 485 634 CIB1 1 217 420 nucleotide interacting region

METHODS

Modular gene expression

Link RBS

CRY2 CIB1

H6

Prefix compatible cloning sites

Link

T Suffix compatible cloning sites [1] Kennedy et al., (2010) Nature Methods 7, 249–252 [2] Meng et al., (2013) Plant Cell 25, 4405-20

Genes coding the A. thaliana N-domain of CRY2 and CIB1 that are required for blue light interaction [1,2] were synthesized by GenScript

RBS myc H6

Different epitope tags

METHODS

Modular gene expression

Link RBS

CRY2 CIB1

H6

Prefix compatible cloning sites

Link

SalI BgIII XhoI BamHI T Suffix compatible cloning sites [1] Kennedy et al., (2010) Nature Methods 7, 249–252 [2] Meng et al., (2013) Plant Cell 25, 4405-20

Genes coding the A. thaliana N-domain of CRY2 and CIB1 that are required for blue light interaction [1,2] were synthesized by GenScript

RBS myc H6

SGGSGGSGGSGG linker sequence BgIII Cre-N SalI XhoI Cre-C BamHI BgIII YFP XhoI BgIII CFP SalI

RESULTS

Modular gene expression

Link RBS

CRY2 CIB1

H6 Link

T

Initial constructs made…..

RBS myc H6

Cre-N Cre-C

T7

Link RBS

CRY2 CIB1

H6 Link

T

RBS myc H6

T7

YFP CFP

AIM1 AIM2

(AmpR) (AmpR) pET16 pET16 SalI BgIII XhoI BamHI NPTII T RFP

T7

loxP

RBS

loxP

RBS

T (ChloR) Cre-recombinase target sits pSB3C Prefix Suffix

RESULTS

Link RBS

CRY2 CIB1

H6 Link

T

RBS myc H6

T7

YFP CFP

AIM 1. Can we detect CRY2-CIB1 function in E. coli?

CRY2-YFP Coomassie stain

M

(kDa) 1

2 1 2 Lys Sol

1- pET16a(+) (control) 2- pETFRET2 Lys – total cellular protein Sol – soluble cell protein CIB1-CFP SDS PAGE sample loading:

75 50 37 25 20 15 10 100

48kDa 86kDa

MycC Ab

1 2 1 2 Lys Sol

Penta-His Ab

1 2 1 2 Lys Sol

Use of the CRY2/CIB1 system may be limited in

• E. coli due to:
• insolubility of CRY2
• proteolysis of CIB1

RESULTS

AIM 1. Can we detect CRY2-CIB1 function in E. coli?

1 2 1 2 Lys Sol 1 2 1 2 Lys Sol

Lets try to express the CRY2 and CIB1 fusion proteins separately and purify them by Immobilized Metal Affinity Chromatography then assay for FRET

Insoluble

(pET28)

85.8 kDa 47.7 kDa CFP T

RBS H6

T7

CRY2

RBS

CIB1

Link H6

YFP

(pET28)

T

T7

Link Link RBS

CRY2 CIB1

H6 Link

T

RBS myc H6

T7

YFP CFP

Soluble 37 kDa fluorescent yellow protein purified

AIM 2. Can we adapt CRY2-CIB1 to trigger gene recombination?

RESULTS

CRY2 CIB1 CREN CREC

rfp

loxP loxP

P

nptII

RFP RFP produced Kanamycin sensitive

Blue light Dark

CRY2

FAD

CIB1

Gene 1

loxP

P

nptII rfp

loxP

NPTII No RFP produced Kanamycin resistant

Link RBS

CRY2 CIB1

H6 Link

T

RBS myc H6

Cre-N Cre-C (AmpR) pET16 NPTII T RFP

T7

loxP

RBS

loxP

RBS

T (ChloR) pSB3C

T7

We co-expressed in E. coli BL21(DE3)

AIM 2. Can we adapt CRY2-CIB1 to trigger gene recombination?

RESULTS

CRY2 CIB1 CREN CREC

rfp

loxP loxP

P

nptII

RFP RFP produced Kanamycin insensitive

• RFP synthesis could be induced as expected by IPTG
• Challenge #1 – unwanted resistance to Kanamycin was observed

in non-induced cells

Serial dilution of culture

LB Media + 10µg/mL Chlor + 50 µg/mL kanamycin

Colony growth RFP RFP + IPTG Colony growth no IPTG

AIM 2. Can we adapt CRY2-CIB1 to trigger gene recombination?

RESULTS

CRY2 CIB1 CREN CREC

rfp

loxP loxP

P

nptII

RFP

Challenge #2 – CRY2-CreN solubility and CIB1-CreC proteolysis (again!)

Coomassie stain

M

(kDa)

75 50 37 25 20 15 10 100

1- pETCre1 2-pET16a(+) (control) SDS PAGE sample loading: 47kDa

(His Ab)

CRY2-CN

1 2 1 2 Lys Sol 1 2 1 2 Lys Sol

His and MycC Ab 70kDa

(MycC Ab)

CIB1-CC

31kDa?

AIM 3. Can we exploit CRY2-CIB1 triggered CRE-recombination for high yield NAD biosynthesis in E. coli?

Not Pursued

Conclusion

Functionality of the light-activated CRY2/CIB1 system may be limited in E. coli due to challenges associated with insolubility of the CRY2-fusion proteins and an apparent sensitivity of the Lysine rich CIB1 protein to proteolysis. Increase CRY2-RP solubility

Slow rate of expression

Promoter, temperature, growth media, alter codon use and [inducer] to slow translation.

Molecular chaperones

Co-express components of protein folding machinery; GroEL/GroES, DnaK, etc..

Possible targets for optimisation by future iGEM teams!! Prevent CIB1-RP cleavage

Determine proteolysis site(s)

Get mass of purified proteins (ESI-MS)

Site direct mutagenesis

Mutate cleavage site residues

Maintain CRY2-CIB1 functionality?

CUSTOMISABLE BLUE-LIGHT SOURCE: A REFERENCE FOR FUTURE TEAMS

• Complete setup cost was $70 AU ($50 US)

Time Intesnsity

PUBLIC OUTREACH

Questacon SciNight - “Good Vibrations” Science in ACTion at the Old Bus Depot Markets

OUTREACH – MELBA COPLAND SECONDARY SCHOOL

black and white perspective ↓ question reasons behind perspective ↓ recognise multiple perspectives

?

iGEM @ ANU

ACHIEVEMENTS

• Started the first ANU iGEM team!
• Proposed a light-controlled system for biosynthesis of toxic metabolites
• Expressed CRY2/CIB1 FRET reporter constructs in E. coli
• Tested solubility and functionality of CRY2/CIB1 FRET reporters
• Expressed Cre-lox recombination reporter in E. coli
• Submitted Cre-lox recombination reporter (BBa_K1750000) and CIB1-YFP reporter

(Part:BBa_K1750001) to Registry

• Reviewed literature of photobioreactor design and biocontainment for industrial scaling (wiki)
• Produced method for simple, cheap, customisable blue-light source
• Ran public and school outreach programs on synthetic biology and ethics

LIGHT vs CHEMICAL INDUCTION

Chemical Light Spatial control and target specificity Low High Temporal control Limited High Cost Expensive Cheap Toxicity Possible None

Kennedy, M.J., Hughes, R.M., Peteya, L.A., Schwartz, J.W., Ehlers, M.D., Tucker, C.L., (2010) “Rapid blue light induction of protein interactions in living cells” Nature Methods 7:973

A new optogenetic device:

• Reversible
• No exogenous ligands required

FUNDING SUPPORT

ACKNOWLEDGEMENTS

Associate Professor Spencer Whitney (ANU Research School of Biology) Dr Colin Jackson (ANU Research School of Chemistry)

Dr Colin Scott (CSIRO) Hafna Ahmed, Jason Whitfield & William Zhang (Jackson Lab, ANU Research School of Chemistry) OUTREACH Alisha Duncan & Natalia Bateman Vargas (ARC Centre of Excellence for Translational Photosynthesis) Simon Mulvaney, Amrita & Julie Harrison (ANU Student Equity) Melissa Easterby & Matt Colbran (Melba Copland Secondary School)

Email: anu.igem@gmail.com Twitter: @anu_igem Wiki: http://2015.igem.org/Team:ANU-Canberra

Thank you – questions? Visit us at Poster 1, Hall C!

Prokaryotic Gene Expression

• Length (up to what length) and show in a diagram
• Lack of post-translational machinery
• Phosphorylation
• Codon bias
• Disulfide-bond formation?
• Liu, National Key Laboratory of Plant Molecular Genetics and

National Center for Plant Gene Research (Shanghai)

• We used eukaryotic systems because used similar to humans.

Link

CFP CRY2

H6 T T7 RBS

85.8 kDa

Link

CIB1 YFP

(47.7kDa) H6 T T7 RBS

P