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

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

Nov 12, 2022 385 likes •645 views

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

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LET THERE BE LIGHT

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Supervisors & Instructors Team Members

Darren Amit Brian Ben Khai Hugh Shahin Cassie Thomas Ranvir Khai Hugh Shahin Cassie Thomas Ranvir

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  • 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

OUR FIRST BIOBRICK: BBa_K1135000

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mKeima : The ‘Unusual’ Fluorescent Protein

  • A monomeric fluorescent beta barrel protein
  • 222 amino acids
  • Large stokes shift
  • 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

mKeima

max excitation ≈ 440 nm (blue) max emission ≈ 620 nm (red)

Large stokes shift

Not just any old fluorescent protein

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Wavelength (nm) Normalised Intensity

400 500 600

eGFP

Excited with blue Emits green

RFP

Excited with yellow Emits red Excited with blue Emits red

mKeima

The difference between peak excitation and emission wavelengths

‘Stokes shift’ of fluorescent proteins

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 250 350 450 550 650

Normalised Light Intensity Wavelength (nm)

mKeima Excitation GFP Excitation mRFP Excitation mKeima Emission GFP Emission mRFP Emission

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  • 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

Genetic manipulation to alter biophysical properties

Piatkevich et al., 2010. JACS 132: 10762.

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  • 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

Construction of BBa_K1135000

Blue excitation Red (620/30nm) filter Blue excitation No filter

Codon Usage Optimizer: Morph Bioinformatics iGEM 2012; Mathews et al., 2007. Current Protocols in Nucleic Acid Chemistry 11. 2. 1-11.

mKeima RNA folding energy

  • 270kcal/mol

hmKeima RNA folding energy

  • 210kcal/mol
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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

eGFP mKeima

autofluorescence

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  • 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 2: Cellular Analysis and Separation

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Blue & Red-light sensing circuit

  • its kind with ‘clean’ wavelength conversion
  • Application 3: A Useful Component of

Genetic Light Circuits

Proof-of-concept idea:

Broaden the ability of light activated circuits

Red-light sensing circuit mKeima mKeima

Wavelength converting module

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Heme PCB

Ho1 PcyA

Background to the Circuit

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

Cph8

Kinase domain (from EnvZ)

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

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0.25 0.5 0.75 1 500 540 580 620 660 700 740 780 Normalized intensity Wavelength, nm

Normalized absorbance acceptor (-) Normalized fluorescence donor (-)

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N O L I G H T B L U E L I G H T

The Circuit

OmpR

P

OmpR

hoI (I15008) PcyA (I15009) constitutive promoter

J23104 mKeima B0034 constitutive promoter B0012 B0011

K1135001

Cph8 constitutive promoter

Cph8

mKeima

  • mpF promoter

(R0082) B0034 B0015 GFP (K208000)

Cph8 Cph8 Cph8

P P

H2O GFP mKeima

Heme PCB

Ho1 PcyA

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Directed Evolution

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

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mKeima mutant #1

Mutagenic PCR

Directed Evolution

Transform Select FACS

#3 and #5 selected

mKeima mutant #2 mKeima mutant #3 mKeima mutant #4 mKeima mutant #5

Test

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A first step is to get people interested…

HUMAN PRACTICES

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Product designer/ Artist (Manuel Wolf)

‘Engineering mKeima’

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Fashion designer (Malene Oddershede Bach)

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Human Practice

Fashion designer (Malene Oddershede Bach)

Print Pattern: Fluorescent-tagged neuroblastoma cells

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‘Spreading the word’ at the BBC

  • A trip to BBC broadcasting house
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Team Photos

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Thank you

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