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)

WHO ARE WE? Biology Engineering Chemistry Mathematics Math humor Stimulus response! Early engineers Early chemists Stimulus response! describe the first Don’t you ever think? dirt molecule

WHO ARE WE? Biology Engineering Chemistry Mathematics Dr Colin Jackson (ANU Research School of Chemistry) A/Prof Spencer Whitney (ANU Research School of Biology)

WHAT IS PHOTO GENE IC ABOUT? optogenetics : the control of cellular dynamics using light International Year of Light

LIGHT-INDUCIBLE CRY2/CIB1 SYSTEM Leaf cell Blue light FAD CRY2 CIB1 CRY2 CIB1 Dark Active Inactive transcription

LIGHT-INDUCIBLE CRY2/CIB1 SYSTEM Yeast, zebra fish, leaf cell FAD CRY2 CIB1 CRY2 CIB1 Blue light EP N EP C Dark Effector protein EP active (EP) inactive • 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.

APPLYING CRY2/CIB1 in E. coli • Ultimate objective – light inducible induction of protein expression for toxic metabolite biosynthesis . Gene 1 Gene 2 Gene 3 Gene 4 Gene - Enzyme1 Enzyme2 Enzyme3 Enzyme4 Product - A B C D Desired product Cell death

APPLYING CRY2/CIB1 in E. coli • Ultimate objective – light inducible induction of protein expression for toxic metabolite biosynthesis . Rate limiting nadB nadA NADB NADA L-aspartate quinolate NAD α -iminoaspartate [NAD] Cell  fitness

APPLYING CRY2/CIB1 in E. coli • Ultimate objective – light inducible induction of protein expression for toxic metabolite biosynthesis . • Proof Of Concept Gaol – high yield NAD biosynthesis nadB nadA NADB NADA L-aspartate quinolate NAD α -iminoaspartate (non-toxic)

APPLYING CRY2/CIB1 in E. coli • Ultimate objective – light inducible induction of protein expression for toxic metabolite biosynthesis . • Proof Of Concept Gaol – high yield NAD biosynthesis nadB nadA NADB NADA L-aspartate NAD

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. Cre-recombinase active FAD CRY2 CIB1 CRY2 CIB1 Blue light CRE N CRE C Dark nadB nadA nadA P P Gene 1 loxP loxP loxP nadB loxP NADB NADA L-aspartate α -iminoaspartate quinolate NAD +

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 ? FAD CRY2 CIB1 CRY2 CIB1 Blue light YFP CFP Dark FRET

EXPERIMENTAL AIMS AIM 1. Can we detect CRY2-CIB1 function in E. coli ? AIM 2. Can we adapt CRY2-CIB1 to trigger gene recombination? FAD CRY2 CIB1 CRY2 CIB1 Blue light CRE N CRE C Dark gene 1 gene 2 2 P P Gene 1 loxP loxP loxP 2 loxP Enz1 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 ? FAD CRY2 CIB1 CRY2 CIB1 Blue light CRE N CRE C Dark nadB nadA nadA P P Gene 1 loxP loxP loxP nadB loxP NADB NADA L-aspartate α -iminoaspartate quinolate NAD +

METHODS Modular gene expression Genes coding the A. thaliana N-domain of CRY2 and CIB1 that are required for blue light interaction [1,2] were synthesized by GenScript 1 485 634 1 217 420 CRY2 CIB1 CRY2 FAD nucleotide interacting region [1] Kennedy et al., (2010) Nature Methods 7 , 249 – 252 [2] Meng et al., (2013) Plant Cell 25, 4405-20

METHODS Modular gene expression Genes coding the A. thaliana N-domain of CRY2 and CIB1 that are required for blue light interaction [1,2] were synthesized by GenScript Prefix Suffix compatible compatible cloning sites cloning sites RBS myc Link RBS Link CIB1 T CRY2 H6 H6 Different epitope tags [1] Kennedy et al., (2010) Nature Methods 7 , 249 – 252 [2] Meng et al., (2013) Plant Cell 25, 4405-20

METHODS Modular gene expression Genes coding the A. thaliana N-domain of CRY2 and CIB1 that are required for blue light interaction [1,2] were synthesized by GenScript Sal I BgI II BamH I Xho I BgI II Xho I Sal I BgI II Cre-N Cre-C YFP CFP Prefix Suffix compatible compatible BgI II Sal I BamH I Xho I cloning sites cloning sites RBS myc Link RBS Link CIB1 T CRY2 H6 H6 SGGSGGSGGSGG linker sequence [1] Kennedy et al., (2010) Nature Methods 7 , 249 – 252 [2] Meng et al., (2013) Plant Cell 25, 4405-20

RESULTS Modular gene expression Initial constructs made….. AIM1 myc Link Link RBS RBS CRY2 YFP CIB1 CFP T (Amp R ) T7 H6 H6 pET16 AIM2 RBS myc Link RBS Link (Amp R ) CRY2 Cre-N CIB1 Cre-C T T7 H6 H6 pET16 Xho I BgI II Sal I BamH I Prefix Suffix loxP RBS loxP (Chlo R ) RFP RBS NPTII T T T7 pSB3C Cre-recombinase target sits

RESULTS AIM 1. Can we detect CRY2-CIB1 function in E. coli ? RBS myc Link RBS Link CRY2 YFP CIB1 CFP T T7 H6 H6 Lys Sol Lys Sol Lys Sol SDS PAGE sample loading: M 86kDa (kDa) 1 2 1 2 1 2 1 2 1 2 1 2 1- pET16a(+) (control) 2- pETFRET2 100 CRY2-YFP Lys – total cellular protein 75 Sol – soluble cell protein CIB1-CFP 50 37 Use of the CRY2/CIB1 48kDa system may be limited in 25 E. coli due to: 20 • insolubility of CRY2 15 • proteolysis of CIB1 10 Coomassie stain Penta-His Ab MycC Ab

RESULTS AIM 1. Can we detect CRY2-CIB1 function in E. coli ? RBS myc Link RBS Link CRY2 YFP CIB1 CFP T T7 H6 H6 Lys Sol Lys Sol 1 2 1 2 1 2 1 2 Lets try to express the CRY2 and CIB1 fusion proteins separately and purify them by Immobilized Metal Affinity Chromatography then assay for FRET Link Link RBS RBS H6 CFP CRY2 T CIB1 YFP H6 T T7 T7 (pET28) (pET28) 85.8 kDa 47.7 kDa Soluble 37 kDa fluorescent yellow Insoluble protein purified

RESULTS AIM 2. Can we adapt CRY2-CIB1 to trigger gene recombination? We co-expressed in E. coli BL21(DE3) myc Link Link RBS RBS (Amp R ) CRY2 Cre-N CIB1 Cre-C T pET16 H6 H6 T7 loxP RBS loxP RFP RBS NPTII T (Chlo R ) pSB3C T T7 FAD CRY2 CIB1 CRY2 CIB1 Blue light CRE N CRE C Dark nptII rfp nptII P P Gene 1 loxP loxP loxP rfp loxP RFP NPTII RFP produced No RFP produced Kanamycin sensitive Kanamycin resistant

RESULTS AIM 2. Can we adapt CRY2-CIB1 to trigger gene recombination? • 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 Colony growth + IPTG CRY2 CIB1 RFP CRE N CRE C Colony rfp nptII P loxP loxP growth no IPTG RFP RFP RFP produced Kanamycin in sensitive LB Media + 10µg/mL Chlor + 50 µg/mL kanamycin

RESULTS AIM 2. Can we adapt CRY2-CIB1 to trigger gene recombination? Challenge #2 – CRY2-Cre N solubility and CIB1-Cre C proteolysis (again!) SDS PAGE sample loading: Lys Lys Sol Sol 1- pETCre1 M 70kDa 1 2 1 2 1 2 1 2 2-pET16a(+) (control) (kDa) (MycC Ab) 100 75 CRY2-C N CRY2 CIB1 AIM 3. Can we exploit CRY2-CIB1 triggered CRE-recombination for CIB1-C C 50 high yield NAD biosynthesis in E. coli ? CRE N CRE C 37 Not Pursued 47kDa 31kDa? (His Ab) rfp nptII P 25 loxP loxP 20 RFP 15 10 Coomassie stain His and MycC Ab

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. Possible targets for optimisation by future iGEM teams!! Determine Slow rate of proteolysis site(s) expression Get mass of purified Promoter, temperature, Increase Prevent proteins (ESI-MS) growth media, alter codon CRY2-RP CIB1-RP Site direct use and [inducer] to slow solubility cleavage mutagenesis translation. Mutate cleavage site Molecular residues chaperones Co-express components of Maintain CRY2-CIB1 protein folding machinery; functionality? GroEL/GroES, DnaK, etc ..

CUSTOMISABLE BLUE-LIGHT SOURCE: A REFERENCE FOR FUTURE TEAMS • Complete setup cost was $70 AU ($50 US) Intesnsity Time

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