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An intergenic complementation approach to engineer a faster fluorescence biosensor Mitesh Agrawal, Jennifer Boothby, Natalie Chilcutt, Joseph Elsherbini, Jennifer Goff Georgia Institute of Technology Americas East Regional Jamboree iGEM 2012

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SLIDE 1

An intergenic complementation approach to engineer a faster fluorescence biosensor

Mitesh Agrawal, Jennifer Boothby, Natalie Chilcutt, Joseph Elsherbini, Jennifer Goff Georgia Institute of Technology Americas East Regional Jamboree iGEM 2012

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SLIDE 2

What is a biosensor?

Signal Biosensor Reporter Sensing machinery Application: Biosensor for Arsenic detection as engineered by Cambridge 2009 iGEM

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SLIDE 3

Quorum Sensing

Signal producing protein Signal receptor protein Signal producing protein

Amount of Autoinducer Signal Binding

LOW CELL DENSITY HIGH CELL DENSITY

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SLIDE 4

GFP Reporter Systems

Traditional Reporter Systems

  • 1. Binding
  • 2. Transcription
  • 3. Translation
  • 4. Accumulation
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SLIDE 5

GFP Reporter Systems

Traditional Reporter Systems Our Novel Reporter System

  • 1. Binding
  • 2. Transcription
  • 3. Translation
  • 4. Accumulation
  • 2. Dimerization
  • 1. Binding
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SLIDE 6
  • 2. Dimerization
  • 1. Binding

Our GFP reporter system

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SLIDE 7

Barnard et. al. 2008

GFP Fragment Reassembly

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SLIDE 8

Agrobacterium tumefaciens

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SLIDE 9

TraR dimerizes only in response to autoinducer signal

TraR monomers TraR-AI complex AI

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SLIDE 10

Adapted from Wilson et. al. 2004

Our approach

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SLIDE 11

Modeling

  • Factors to consider:

1) Time taken for Diffusion of Auto-Inducer across cell membrane (T1) 2) Time taken for binding of AI to the TraR protein (T2) 3) Time required for transcription + translation + accumulation (T3) 4) Time Required for the dimerization rate of GFP (T4) 5) 5000-10000 GFP molecules are required per cell for it be detected

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SLIDE 12

T1 – Diffusion of autoinducer into the cell

T1 calculated will be similar in both the current GFP Reporter system and our proposed GFP Reporter system. Traditional System Novel System

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SLIDE 13

Number of GFP-TraR fusion proteins (i) based on TraR half-life

𝑒𝑗 𝑒𝑒 = 𝑄𝑠𝑝𝑛𝑝𝑒𝑓𝑠 𝐡𝑑𝑒𝑗𝑀𝑗𝑒𝑧 βˆ’ πΈπ‘“π‘•π‘ π‘π‘’π‘π‘’π‘—π‘π‘œ 𝑆𝑏𝑒𝑓. (𝑗)

  • i = number of GFP-TraR fusion proteins
  • Degradation Rate = ln2/ t1/2
  • t1/2 of TraR ranges from 3-680 minutes
  • Condition: The fusion proteins have reached steady state

𝒋 = 𝑸 βˆ— π’–πŸ/πŸ‘ π’Žπ’πŸ‘

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SLIDE 14

Number of GFP-TraR fusion proteins based

  • n TraR half-life

Steady State Value of Fusion protein ranges from 100 – 23000 molecules/cell

*The plot here shows the linear dependence of i vs TraR half-life ranging from 212-680 minutes

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SLIDE 15

T2 – Binding of auto-inducer to receptor

Traditional System Novel System Modeled along

  • Receptor-Ligand Kinetics

Binding Rate Unoccupied Receptors Auto-inducer AI+TraR fusion complex Dissociation Rate

  • Calculated T2 = 130

seconds

  • Similar in both cases
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SLIDE 16

T3 – Transcription and translation

Transcription Translation

  • Our reporter system

provides an advantage

  • ver the old Reporter

system

  • Absence of

Transcription+Translation after the addition of AI

  • The TraR-GFP fusion

proteins are already accumulated at a steady state value (i)

  • T3 = 0 for our system

Traditional System

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SLIDE 17

T3 – Accumulation of protein

Traditional system

Accumulation

  • For the old Reporter system, it has

been demonstrated that T3 = 1-3 hours

  • To establish consistency of our

model with experimental results, we modeled T3 for old system: T3 (modeled) = 0.5-2 hours

  • z= number of GFP protein ;

u= growth rate of bacteria; D= degradation rate of GFP

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SLIDE 18

T5 – Dimerization of TraR-GFP fragment fusions

Novel system T5 = 3-200 seconds depending on the steady state value

  • f GFP-TraR
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SLIDE 19

Total time taken/Conclusion

Traditional GFP Reporter System Our novel GFP Reporter System T1=Similar T1=Similar T2= Similar T2= 2-3 mins = Similar T3= 60-180 mins T3= 0 (Already accumulated) T4=0 (non-existent) T4= 3-200 seconds Total time= 1-3 hours = 60-180 mins Total Time= 2-6 mins

  • Thus, our model represents that our novel GFP reporter system reduces the

time required for GFP expression by a factor of 30, supporting our hypothesis.

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SLIDE 20

Results

Leucine Zipper Controls

zipper-NGFP alone zipper-CGFP alone NGFP and CGFP

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SLIDE 21

Results

  • We successfully cloned TraR-NGFP and TraR-

CGFP Fusions

  • We were unable to see fluorescent colonies on

plates under many different conditions

– Different concentrations of autoinducer – Different methods of induction – Growth at different temperatures

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SLIDE 22

Flow Cytometry – No GFP induction

Cell Count Fluorescence Level

No induction of GFP fragments

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SLIDE 23

Flow Cytometry – Leucine Zippers

Cell Count Fluorescence Level

Leucine zipper controls

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SLIDE 24

Flow Cytometry – TraR-GFP fusions

Frequency Fluorescence Level

Experimental TraR-GFP fusions

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SLIDE 25

Results- Flow Cytometry

Cell Count Fluorescence Level

Experimental TraR-GFP fusions

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SLIDE 26

BioBricks Submitted

Bba_K916001 TraR-NGFP Bba_K916000 TraR Bba_K916002 TraR - CGFP Bba_K916003 Leucine Zipper- NGFP Bba_K916004 Leucine Zipper- CGFP

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SLIDE 27

BioBricks Submitted

Bba_K916001 TraR-NGFP Bba_K916000 TraR Bba_K916002 TraR - CGFP Bba_K916003 Leucine Zipper- NGFP Bba_K916004 Leucine Zipper- CGFP

* *

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SLIDE 28
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SLIDE 29

Characterizing the Las receiver BBa_K091134

  • Designed by Davidson-Missouri Western 2008 iGEM

team

  • Contains the LasR receptor from the pathogenic

bacterium Pseudomonas aeruginosa with a GFP reporter

  • Traditional quorum-sensing-based biosensor
  • How part should function:

– Produce LasR in the presence of IPTG – LasR binds to autoinducer – LasR + autoinducer binds to promoter – Transcription is reported by GFP expression

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SLIDE 30

Sequencing of Part

  • The PLlac 0-1 promoter which controls the

expression of lasR is completely absent.

  • In place of the promoter is other P. aeruginosa

sequence

  • All other components of the part are as reported
  • Given the lack of the promoter for lasR expression,

we anticipated that a fluorescent signal will not be produced in the presence of autoinducer

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SLIDE 31

Testing of LasR Receiver

  • Transformed the

part into E. coli

  • When the cells

were grown in the presence of autoinducer and IPTG, no fluorescent signal was observed.

IPTG -

+ +

AI

  • +
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SLIDE 32

Suggestions for Improving LasR Receiver

  • Replace incorrect sequence upstream of

lasR with the correct promoter (PLlac 0-1)

  • As the rest of the part appears to be as

reported, this should restore the proper function to the part

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SLIDE 33

Human Practices

http://www.forsythnews.com/m/section/3/article/14721/

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SLIDE 34

Establishing New iGEM teams

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SLIDE 35

Conclusion

  • We submitted 5 well-characterized and

documented BioBricks

  • We have a working mathematical model

with which to test our system

  • We have reason to believe that our system

can work with further improvements

  • We characterized another team’s part and

improved its documentation

  • We helped to seed the first iGEM high

school team in Georgia

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SLIDE 36

Acknowledgements

  • Dr. Clay Fuqua – University of Indiana
  • Dr. Lynne Regan – Yale University

UROP MS&T MoNaCo (NSF grant 1110947) School of Biology PURA