LET THERE BE LIGHT Supervisors & Instructors Amit Darren - PowerPoint PPT Presentation

LET THERE BE LIGHT

Supervisors & Instructors Amit Darren Brian Ben Team Members Khai Shahin Khai Ranvir Cassie Thomas Thomas Shahin Ranvir Cassie Hugh Hugh

OUR FIRST BIOBRICK: BBa_K1135000 • In keeping with iGEM philosophy “Get & Give & Share” • A novel component for biological machines • A biological wavelength converting module → diverse applications • Conforms to iGEM standards and deposited into parts registry – available for use

mKeima : The ‘Unusual’ Fluorescent Protein A monomeric fluorescent beta barrel protein • 222 amino acids • mKeima Large stokes shift Large stokes shift • max excitation ≈ 440 nm (blue) Not just any old max emission ≈ 620 nm (red) fluorescent protein Originally developed for cross correlation spectroscopy • Advantages: • Decreased signal: noise in cells compared to GFP • Enhanced accuracy in quantification of multiple • fluorescent protein reporters Kogure et al., 2006. Nat Biotech 24:577; Henderson et al., 2009. JACS 131: 13212

‘Stokes shift’ of fluorescent proteins 1 Normalised Light Intensity 0.9 0.8 RFP eGFP mKeima 0.7 0.6 Excited with blue Excited with blue 0.5 Normalised Intensity 0.4 Emits green 0.3 Excited with yellow 0.2 Emits red Emits red 0.1 400 500 600 0 Wavelength (nm) 250 350 450 550 650 Wavelength (nm) The difference between peak excitation and emission mKeima Excitation GFP Excitation mRFP Excitation wavelengths mKeima Emission GFP Emission mRFP Emission

Genetic manipulation to alter biophysical properties • Excited state proton transfer (ESPT) relay mechanism of mKeima long stokes shift • Genetics can alter chemistry! • ‘Genetic stokes shifts! ‘ • e.g. mCherry stokes shift variants by altering ESPT Piatkevich et al., 2010. JACS 132: 10762.

Construction of BBa_K1135000 • Codon optimised for mammalian cell expression • Collaboration with Dr. Martin Pule (Clinical research fellow, UCL CI) • Gene sequence expresses well in bacteria too • We aimed to make some tools using the bacterial expression biobrick mKeima RNA folding energy hmKeima RNA folding energy -270kcal/mol -210kcal/mol Blue excitation Blue excitation No filter Red (620/30nm) filter Codon Usage Optimizer: Morph Bioinformatics iGEM 2012; Mathews et al., 2007. Current Protocols in Nucleic Acid Chemistry 11. 2. 1-11.

Application 1: Glucose Sensor • mKeima for in vitro diagnostics • Avoid autofluorescence background from samples • Proof of concept: glucose sensor (pCSTA promoter + reporter) • Biobrick BBa_K1135004 constructed: pCSTA promoter — RBS – mKeima – terminator • Compared against reporters GFP and RFP • A more reliable biosensor than these previous biobricks mKeima eGFP autofluorescence

Application 2: Cellular Analysis and Separation • Collaboration with Arnold Pizzey (flow cytometrist) • Flow cytometric sorting FACS separation of mKeima from GFP based on emission from RFP based on excitation • Tool for research into cancer, stem cells, gene therapy

Application 3: A Useful Component of Genetic Light Circuits • its kind with ‘clean’ wavelength conversion Broaden the ability of light activated circuits Proof-of-concept idea: • • mKeima • Red-light Blue & Red-light sensing circuit sensing circuit • • mKeima • Wavelength converting module •

Background to the Circuit Cph8 is a fusion protein which was originally used in bacterial photography Red light Kinase domain sensing domain (from EnvZ) (from Cph1) Cph8 Phycocyanobilin (PCB) is the chromophore of the light sensing domain. PCB is produced from heme by the enzymes Ho1 and PcyA Ho1 PcyA Heme PCB

Absorbance of Cph8 Emission of mKeima Overlap !!! (^_^)

1 Normalized absorbance acceptor (-) Normalized fluorescence donor (-) 0.75 Normalized intensity 0.5 0.25 0 500 540 580 620 660 700 740 780 Wavelength, nm

K1135001 The Circuit B0034 B0012 B0011 J23104 mKeima Cph8 constitutive constitutive hoI PcyA constitutive promoter promoter (I15008) (I15009) promoter mKeima Ho1 PcyA Cph8 Heme PCB mKeima B N L Cph8 P O U B0015 B0034 E H 2 O ompF promoter GFP (R0082) (K208000) L Cph8 I L P OmpR G I H G T H P GFP Cph8 OmpR T

Directed Evolution Mutagenic PCR 5 1 Clone into Amplification light sensing circuit 2 4 Selection Transformation 3

Directed Evolution Mutagenic PCR Select Transform Test mKeima mutant #1 FACS mKeima mutant #2 mKeima mutant #3 mKeima mutant #4 mKeima mutant #5 #3 and #5 selected

HUMAN PRACTICES A first step is to get people interested…

‘ Engineering mKeima ’ Product designer/ Artist (Manuel Wolf)

Fashion designer (Malene Oddershede Bach)

Print Pattern: Human Practice Fluorescent-tagged neuroblastoma cells Fashion designer (Malene Oddershede Bach)

‘Spreading the word’ at the BBC • A trip to BBC broadcasting house

Team Photos

Thank you