MELAN-OMA-GOSH! Harnessing Yarrowia lipolytica for early detection of - - PowerPoint PPT Presentation

▶
melan oma gosh
SMART_READER_LITE
LIVE PREVIEW

MELAN-OMA-GOSH! Harnessing Yarrowia lipolytica for early detection of - - PowerPoint PPT Presentation

Mar 23, 2023 191 likes •544 views

MELAN-OMA-GOSH! Harnessing Yarrowia lipolytica for early detection of cancer Hendrix Arkansas Detecting Melanoma through an Alkane-Based Biosensor Why is cancer an Background important problem? How is the problem currently addressed?

slide-1
SLIDE 1

MELAN-OMA-GOSH!

Harnessing Yarrowia lipolytica for early detection of cancer Hendrix Arkansas

slide-2
SLIDE 2

Background

Detecting Melanoma through an Alkane-Based Biosensor

  • Why is cancer an

important problem?

  • How is the problem

currently addressed?

  • How do we plan to

address the problem?

  • Where did we get our

idea?

  • How will our machine

work?

slide-3
SLIDE 3

American Cancer Society. 2014. “Cancer facts & Figures 2014.” www.cancer.org/research/cancerfactsstatistics/cancerfactsfigures2014/index

Why is combating cancer important?

  • Cancer is a leading cause of death world wide.
  • Most common form of cancer in the U.S. is skin

cancer.

  • The most deadly form of skin cancer is melanoma.
  • Late stage melanoma 5 year survival rate = 15%.
  • With detection and treatment before metastasis,

5 year survival rate = 99%.

slide-4
SLIDE 4

MELANOMA & ARKANSAS

National Cancer Institute & CDC. 2014. “State Cancer Profiles- Arkansas.” http://statecancerprofiles.cancer.gov/recenttrend/index.php?0&05&0&9599&001&999&00&0&0&0&1#results

slide-5
SLIDE 5

CURRENT MELANOMA DETECTION

Adapted from American Academy of Dermatology. 2014. “Melanoma: Signs and Symptoms.” https://www.aad.org/dermatology-a-to-z/diseases-and-treatments/m---p/melanoma/signs-symptoms

slide-6
SLIDE 6

Crowley, A. 2013. “How my beloved dog found my cancer” http://www.telegraph.co.uk/lifestyle/pets/9935073/How-my-beloved-dog-found-my-cancer.html

slide-7
SLIDE 7

HOW DO DOGS DO IT?

Volatile compounds

slide-8
SLIDE 8

HOW DO DOGS DO IT?

Volatile compounds

DETECTION

slide-9
SLIDE 9

Abaffy T. et al. 2011. “A case report - Volatile metabolomic signature of malignant melanoma using matching skin as a control” Journal of Cancer Science Therapy 3(6): 140–144.

MELANOMA PRODUCES VOLATILE COMPOUNDS

slide-10
SLIDE 10

INTRODUCING YARROWIA LIPOLYTICA

  • Non-conventional yeast
  • Used in industrial applications
  • Efficient secretory

pathway

  • Useful for Synthetic Bio

applications

  • Able to use alkanes as sole

carbon source

  • Inducible
  • Only works in the

absence of other carbon sources

slide-11
SLIDE 11

ALKANE RESPONSE IN Y. LIPOLYTICA

1Yamagami S. et. Al (2004). “A Basic Helix-Loop-Helix Transcription Factor Essential

for Cytochrome P450 Induction in Response to Alkanes in Yeast Yarrowia lipolytica” Journal of biological chemistry (279:22183-22189.)

slide-12
SLIDE 12

Approach

Detecting Melanoma through an Alkane-Based Biosensor

  • What is the

structure of our construct?

  • What are our

experimental goals?

  • How do we address

those goals?

slide-13
SLIDE 13

WHAT IS THE STRUCTURE OF OUR CONSTRUCT?

pBR322 Docking

Leu2

  • Y. lipolytica expression vector

K592009

slide-14
SLIDE 14

2014 EXPERIMENTAL GOALS

Reporter Testing Part Generation

Assemble & Test Biosensor

slide-15
SLIDE 15

CEN6/ARSH4

  • rigin

antibiotic resistance

K592009 Gal 1 CYC1t

slide-16
SLIDE 16
slide-17
SLIDE 17
slide-18
SLIDE 18

Gal 1 K592009

CEN6/ARSH4

Gal 1 K592009

CEN6/ARSH4

Gal 1 CYC1t K592009

CEN6/ARSH4

Gal 1 CYC1t K592009 Blue

slide-19
SLIDE 19

ORIENTATION SCREEN

M 1 2 3 4 5 700bp - Agarose gel electrophoresis

slide-20
SLIDE 20

COLORING S. CEREVISIAE

~72 hours growth

SC-URA (GLU) SC-URA (GAL) Negative control Positive Control

slide-21
SLIDE 21
slide-22
SLIDE 22

FLUORESCENT PROTEINS

Dissection microscope with fluorescent capabilities Control P416-Gal1

  • S. cerevisiae

p416-Gal1GFP White Light UV Light

~24 hours of growth!

slide-23
SLIDE 23

FLUORESCENT PROTEINS

Fluorescent microscopy Control p416-Gal1

  • S. cerevisiae

p416-Gal1 GFP White Light UV Light + Red Filter

~24 hours of growth!

  • S. cerevisiae

p416-Gal1 RFP UV Light + Green Filter

slide-24
SLIDE 24

pBR322 Docking

Leu2

  • Y. lipolytica expression vector

RFP/GFP

PARTS

slide-25
SLIDE 25

3 x ARE1

167bp, too big for oligo synthesis

E X

slide-26
SLIDE 26

3 x ARE1 divided into 2 parts 3 x ARE1 E X E X

slide-27
SLIDE 27

2 parts divided into 4 oligos 3 x ARE1 E X E X E X

slide-28
SLIDE 28

3 x ARE1 pSB1C3

pSB1C3

  • rigin

antibiotic resistance

  • rigin

E X P S antibiotic resistance E X P S

pSB1C3

slide-29
SLIDE 29

In Summary

Reporter Gene Testing

  • S. Cerevisiae is able to

express the blue chromoprotein and both RFP and GFP

  • Blue chromoprotein

expression seen after 72 hrs.

  • RFP & GFP expression

seen after 24 hrs.

Part Construction

  • All parts transformed

into pSB1C3 plasmid backbone.

  • Verified by restriction

analysis.

  • Awaiting sequence

verification.

slide-30
SLIDE 30

Detecting Melanoma through an Alkane-Based Biosensor

  • Reporter time course.
  • Assemble device via

biobrick assembly.

  • Clone device into Y.

Lipolytica expression vector and transform into yeast

  • Test Construct.

Future Directions

slide-31
SLIDE 31

TESTING STRATEGIES

Soak paper in volatile hydrocarbons Culture Y. lipolytica upside down on media lacking carbon source

slide-32
SLIDE 32

Implications

Detecting Melanoma through an Alkane-Based Biosensor

slide-33
SLIDE 33
slide-34
SLIDE 34

ATTRIBUTIONS

Team Members: Noah Beggs Seana Corbin Chase Davis Krystal Kim Alex McIntyre Jordan Spennato Jay Stanley Faculty Advisor:

  • Dr. Richard Murray

Collaborators:

  • Dr. A. Caro
  • Dr. A. Duina
  • Dr. A. Schurko

Funding: Hendrix Odyssey Sponsors: BioBasic