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Fluidic Platforms FOR High-Throughput Protein Synthesis AND Their Applications IN Drug Screening

Permanent Link: http://ufdc.ufl.edu/UFE0041542/00001

Material Information

Title: Fluidic Platforms FOR High-Throughput Protein Synthesis AND Their Applications IN Drug Screening
Physical Description: 1 online resource (157 p.)
Language: english
Creator: Khnouf, Ruba
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: cell, drug, expression, free, high, microfluidics, protein, screening, throughput
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Protein expression and purification are a limiting step in many areas of research including structural biology, drug screening, and protein function and interaction studies. Conventional protein expression techniques are laborious and time consuming. In order to overcome these obstacles scientists have developed multiple formats and approaches for cell-free protein synthesis, however, in spite of these developments conventional methods are still prevalent mainly because of their higher protein synthesis yield and lower cost. To address these two challenges microfluidics based platforms were tested and developed to enhance protein synthesis yield, miniaturize the reaction volume, lower reagent consumption and cost, and allow high throughput screening. Two microfluidic devices were tested and conditions were optimized to both miniaturize cell-free protein synthesis and to enhance its yield. In the first device the passive pumping mechanism was utilized to provide cell-free protein synthesis reactions with nutrients and energy for yield enhancement. Different parameters were investigated to optimize expression yield and reagent consumption. In the second device, miniaturization of continuous exchange cell-free protein synthesis was optimized; nutrients and energy components were supplied to the reaction through a semipermeable membrane. The device was also designed to be compatible with commercial detection and dispensing systems. High throughput protein synthesis was demonstrated by expressing multiple proteins that can be optically detected. The versatility of the device was shown by expressing both soluble and insoluble membrane proteins that are functional and can be further used in assays such as drug screening. Potential applications of the platforms in drug screening were also demonstrated by searching for ?-lactamase inhibitors, important components of modern antibiotics. Drug screening was further demonstrated by utilizing cell-free protein synthesis for detecting protein synthesis inhibitors and their molecular targets. Results show the versatility of the device and the potential of using fluidic platforms for the advancement of protein expression and drug screening.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ruba Khnouf.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Fan, Zhonghui H.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041542:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041542/00001

Material Information

Title: Fluidic Platforms FOR High-Throughput Protein Synthesis AND Their Applications IN Drug Screening
Physical Description: 1 online resource (157 p.)
Language: english
Creator: Khnouf, Ruba
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: cell, drug, expression, free, high, microfluidics, protein, screening, throughput
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Protein expression and purification are a limiting step in many areas of research including structural biology, drug screening, and protein function and interaction studies. Conventional protein expression techniques are laborious and time consuming. In order to overcome these obstacles scientists have developed multiple formats and approaches for cell-free protein synthesis, however, in spite of these developments conventional methods are still prevalent mainly because of their higher protein synthesis yield and lower cost. To address these two challenges microfluidics based platforms were tested and developed to enhance protein synthesis yield, miniaturize the reaction volume, lower reagent consumption and cost, and allow high throughput screening. Two microfluidic devices were tested and conditions were optimized to both miniaturize cell-free protein synthesis and to enhance its yield. In the first device the passive pumping mechanism was utilized to provide cell-free protein synthesis reactions with nutrients and energy for yield enhancement. Different parameters were investigated to optimize expression yield and reagent consumption. In the second device, miniaturization of continuous exchange cell-free protein synthesis was optimized; nutrients and energy components were supplied to the reaction through a semipermeable membrane. The device was also designed to be compatible with commercial detection and dispensing systems. High throughput protein synthesis was demonstrated by expressing multiple proteins that can be optically detected. The versatility of the device was shown by expressing both soluble and insoluble membrane proteins that are functional and can be further used in assays such as drug screening. Potential applications of the platforms in drug screening were also demonstrated by searching for ?-lactamase inhibitors, important components of modern antibiotics. Drug screening was further demonstrated by utilizing cell-free protein synthesis for detecting protein synthesis inhibitors and their molecular targets. Results show the versatility of the device and the potential of using fluidic platforms for the advancement of protein expression and drug screening.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ruba Khnouf.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Fan, Zhonghui H.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041542:00001


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Full Text

PAGE 16

1.1 Protein Production and Purification

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1.1.1 Cell -F ree Protein Synthesis (CFPS)

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1.1.1.1 Continuous exchange cell-free (CECF) protein s ynthesis

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1.1.1.2 Advantages of CECF

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1.1.1.3 CECF f ormats

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1.1.2 Protein Purification

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1.1.3 High Throughput Protein Synthesis (HTPS)

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1.2 Mi croFluidics

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1.3 Miniaturization and Biotechnology

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1.4 Application s of M icrofluidics and BioMEMS

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1.5 Drug S creening and High -Throughput Screening

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1.5.1 Optical Detection for Drug Screening

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1.5.2 Enzyme Fingerprinting and Screening

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1.5.3 Membrane Proteins 1.5.3.1 Nanolipoproteins (N LP)

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1.5.3.2 Membrane protein expression in cell-free systems and its advantages

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1.5.4 Antibiotic Resistance

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1.5.5 B-L actamase

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1.5.6 Protein Synthesis Inhibitors (PSI)

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1.6 Motivation and Objectives

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Nat. Methods 3

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," 2.1 Introduction

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2.2 Materials and Methods 2.2.1 Materials 2.2.2 Device Fabrication

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2.2.3 Protein Expression 2.2.4 Luciferase Detection

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2.2.5 Applying Passive Pumping to Cell-Free Protein Expression

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2.3 Results and Discussion 2.3.1 Protein Expression in Device

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2.3.2 Amount of Nutrient Solution

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2.3.3 Feeding Frequency of Nutrient Solution and Its Delivery Rate

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2.3.4 Amount of Reaction Mix 2.4 Conclusion

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0 2 4 6 8 10 12 14 16 18 20Luminescence (x10 6RLU/s)Axis TitleDevice 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 1 2 3 4 5Luminescence (106, RLU/s)Amount of Nutrient Solution ( l)

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Luminescence (10 6RLU/s) Luminescence (106RLU/s)

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3.1 Introduction

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3.2 Materials 3.3 Methods 3.3.1 Prototype Device Fabrication

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3.3.2 Protein Expression 3.3.3 Luciferase Detection

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3.3.4 Membranes 3.3.5 Reaction Mix and Feeding Solution Volume Ratio

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3.3.6 Exchange Surface Area of Membrane

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3.3.7 96 Well Plate Design

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3.3.8 PDMS Thickness 3.3.9 Device Fabrication 3.3.10 Device T ests

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3.3.10.1 The dispensing test 3.3.10.2 Luminescence measurement 3.3.11 Protein Purification

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3.4 R esults 3.4.1 Membrane Effect

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3.4.2 Reaction Mix and Feeding Solution Ratio

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3.4.3 Membrane Surface Area 3.4.4 PDMS for Microstamping

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3.4.5 Device Fabrication and Testing

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3.4.6 Protein Purification 3.5 Conclusion

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0 1000000 2000000 3000000 4000000 5000000 6000000 7000000Luminescence (RLU/s)ve Spectrum Disk Millipore Spectrum Sheet GE

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0 2000000 4000000 6000000 8000000 10000000 12000000 14000000 16000000 18000000Luminescence (RLU/s) ve 100 150 200 300

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1 10 100 1000 10000 100000 1000000 10000000 100000000 456789 10Luminescence (RLU/s)Membrane Surface Area (mm^2)

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0 10 20 30 40 50 60 70 0 5001000150020002500Thickness (um)Speed (RPM) 2 cm 4 cm 0 5 10 15 20 25 30 35 0 5001000150020002500Thickness (um)Acceleration (RPM2) 2cm 4cm

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0 2000000 4000000 6000000 8000000 10000000 12000000 14000000 16000000 18000000Luminescence (RLU/s) negative Tube Tube with FS Device 0 100000 200000 300000 400000 500000 600000 700000 800000Luminescence (RLU/s) Fabricated Commercial

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0 500 1000 1500 2000 2500 3000 3500 4000Luminescence (RLU/s) ve rxn mix

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4.1 Introduction

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4.2 Materials and Methods 4.2.1 Plasmid C onstruction and Expression

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4.2.2 Protein Expression

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4.2.3 Detection of G lucoronidase (GUS)

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4.2.4 B-Galactosidase (LacZ) D etection 4. 2.5 Alkaline Phosphatase (AP) Detection

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4.2.6 BLactamase (B -Lac) D etection 4.3 Results 4.3.1 Luciferase Expression Yield

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4.3.2 Protein Expression Levels

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4.3.3 Co -Expression 4.4 C onclusion

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A B C

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1 2 3 4 5 6 7

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0 100000000 200000000 300000000 400000000 500000000 600000000 700000000 0 20406080100120Fluorescence (FLU/s)Time (minutes) Background GUS 0 100000000 200000000 300000000 400000000 500000000 600000000 700000000 800000000 900000000 1E+09 05 101520253035Fluorescence(FLU)Time (minutes)

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0 0.2 0.4 0.6 0.8 1 1.2 1.4 02468 10121416Absorbance(AU)Time ( mins ) lactamase luciferase

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0 0.2 0.4 0.6 0.8 1 1.20 2 4 6 8Normalized Expression Yield (AU)Time (Hours) Device Regular Well

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0 0.2 0.4 0.6 0.8 1 1.2Normalized FluorescenceA 0 0.2 0.4 0.6 0.8 1 1.2 1.4Normalized Fluorescence 0 0.2 0.4 0.6 0.8 1 1.2Normalized Luminescence (AU) 0 0.2 0.4 0.6 0.8 1 1.2Normalized Fluorescence (AU)D 0 0.2 0.4 0.6 0.8 1 1.2 1.4Normalized Fluorescence (AU) 0 0.2 0.4 0.6 0.8 1 1.2Normalized Absorbance (AU) B C E F

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9163648 204770 92749 44.75 50216325 578202 567482 86.85 59381261 1133778 427 52 404715 142080 42365 2.85 56818101 39955264 21631131

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0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1A 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1B 0 0.2 0.4 0.6 0.8 1C 0 0.2 0.4 0.6 0.8 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0 0.2 0.4 0.6 0.8 1 D E F

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881021 43952 92749 20.05 14483424 585477 567483 24.74 19846715 225067 427 88.18 238355 43163 42365 5.52 45474453 33927339 21631131 1.34

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5.1 Introduction

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5.1.1 Clavulanate A cid 5.1.2 Sulbactam

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5.1.3 Tazobactam

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5.2 Materials and Methods 5.2.1 Membrane Protein Expression 5.2.1.1 Vesicle and retinal p reparation

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5.2.1.2 Protein expression 5.2.1.3 SDS PAGE and W estern b lotting

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5.2.2 BLactamase Inhibition A ssays

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5.2.3 Protein Synthesis Inhibitor Target Selection and Design

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5.3 Results and Discussion 5.3.1 Miniaturization of the Membrane Protein Expression A ssay

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5.3.2 Membrane Proteins Solubility

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5.3.3 Lactamase Inhibitor Detection as an Example for Drug Screening

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5.3.4 PSI Target S creening

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5.4 Conclusion

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A B

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A B C D

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0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 02468 1012Absorbance (AU)Time (minutes) Negative Positive 25.6 mg/ml 2.56 mg/ml .256 mg/ml

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0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 02468 1012Absorbance (AU)Time (minutes) Negative Positive 3.2 mg/ml .32 mg/ml

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20 0 20 40 60 80 100 120 3.5 2.5 1.5 0.50.51.52.5% InhibitionConcentration log( g/ l)Clavulanate Acid Tazobactam Sulbactam Cefotaxime

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A D C B

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0.2 0 0.2 0.4 0.6 0.8 1 1.2 1 10100100010000Fraction of expressionAmount of taget RNA added (log equivalent moles to Ricin) Sp PM IA NL

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0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 0.01 0.1 1 10 100Luminescence signal (RLU/s)Amount of Hygromycin B RNA target (equivqlent Moles) 0 10000000 20000000 30000000 40000000 50000000 60000000 70000000 80000000 90000000 100000000 0.010.1 1 10100Luminescence Signal (RLU/s)Amount of sparsomycin target (log equivalent moles) Sparso Target HB1 Target

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6.1 Assessment o o o

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6.2 Future Work 6.2.1 Evaporation C ontrol

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6.2.2 In Situ Protein Purification

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6.2.3 Automated Dispensing and Mixing

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6.2.4 Universal Detection Method

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