[PPT] - Renal Failure 1 1 National Yang Ming University 2 Chronic Renal PowerPoint Presentation

SLIDE 1

1

For Improving the Quality of Life of Patients Suffering from Chronic Renal Failure A Bacteria-based Micro-dialysis Machine

SLIDE 2

2

National Yang Ming University

SLIDE 3

3

Chronic Renal Failure

 Kidneys no longer remove metabolic wastes and maintain balance of body fluid efficiently. Costing  Inconvenient.  Constrictions on diets.  Injuries of blood vessel, ultimately causing unrecoverable damage.

Taiwan Broadcasting System (TBS) People Post (PeoPo) http://media.peopo.org/fullscreen.htm?v= 76 28 fe 54 &auto=true http://www.onemedplace.com/blog/wp- content/uploads/2008/05/soft_case_for_brochure.jpg

SLIDE 4

4

Our Solution

Traditional Hemodialysis BacToKidney Ability to remove wastes Portable X Time Saving X Self-regulation X

SLIDE 5

5

Our BacToKidney!!

pH sensor Attachment Waste removal Time regulation

SLIDE 6

6

The pH Sensor

pH

SLIDE 7

7

Why Need pH Sensor? Motivation

When our system arrives in the intestine (of high pH condition, around pH 7-8), it senses the pH condition and starts to work. In this subsystem, we are going to create a pH sensor which senses high pH.

Goal

To create a pH sensor that can sense high pH condition and start gene expression.

pH

SLIDE 8

8

How to Sense pH?

NhaA NhaR H+

Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+

Na+

H+ H+

Na+ pH Derived as pH sensor pNhaA RBS nhaA

SLIDE 9

Attachment

Attach

SLIDE 10

How to attach?

Lipoprotein signal peptide (lpp)

Targeting the protein to the outer membrane, facing the periplasm.

Outer membrane protein (ompA)

Crossing the outer membrane of E. coli.

Fimbriae protein H (fimH) Mediating attachment between cell and bacteria

pNhaA

fimH

mpA

lpp

RBS

ter ter

Attach

SLIDE 11

FimH-mediated attachment  Subunit of Type I fimbriae  Express on the surfaces of all E. coli  Receptor and Ligand: FimH (Receptor binding domain) & Mannose

FimH

Attach

SLIDE 12

How to detach?

Time-regulated Protease expression Protease

Detach ter

protease

RBS

ter Attach

mpA

RBS fimH

lpp

Protease cleavage site

ter ter

pNhaA Attach (Time-regulated)

SLIDE 13

13

Time Regulation

Time

SLIDE 14

14

When to Detach? Attachment Detachment time Timer

Timing Time

SLIDE 15

15

Starter Oscillatory System

The Timer

1. Timer starts on a signal from the pH sensor.
2. “Count” time using an oscillator.
3. Stop the timer after the specified amount of

time has passed.

Input Output time pH 7 to 8 Stopper AND

Time

SLIDE 16

16

Stopping the Timer

 Protein produced by the oscillatory system accumulates.  After the threshold is reached, the output promoter of the timer suddenly increases its activity.

Accumulated protein amount time Accumulated protein amount versus time

Threshold

time Output promoter Activity Output promoter activity versus time

Threshold

Time

SLIDE 17

17

The Oscillatory System

Starter Stopper Input AND Output Oscillatory System Cyanoxilator Reloxilator

Oscillatory System

Output Protease Time

SLIDE 18

18

The Cyanoxilator

(The oscillator with the longer period)

Cyanobacterial Oscillator (Cyanoxilator)

A natural oscillator occurring in Cyanobacteria. Has a period of 14 - 60 hrs.

Output KaiA KaiC Oscillator KaiB SasA RpaA

P P P P P P P P P P P P P P P P P P

Protease Time

SLIDE 19

19

The Reloxilator

(The oscillator with the shorter period)

Relaxation Oscillator (Reloxilator)

A tuneable synchronized oscillator. Uses parts from: l phage, V. fisheri, and E. coli. Has a period of around 45 mins.

Output HtlB CII CIIICd CII Oscillator LuxI LuxR Tuner Synchronizer Protease Time

SLIDE 20

20

Urea & Guanidine Removal

Waste Removal

SLIDE 21

21

Why Remove Urea and Guanidine?

Waste Removal

SLIDE 22

22

How to Remove Urea & Guanidine?

 Absorption by bacteria (1) Urea transporter (2) Guanidine transporter

Waste Removal

SLIDE 23

23

Active transporter Passive transporter G U G U G U U U U

Absorption of Urea & Guanidine (intestinal lumen) (intestinal cells) (blood)

U U U U U U U U G U U G G G U G G Waste Removal

SLIDE 24

24

pUreD yaaU ureI

ureR ureR ureR

ureR U U G G U U G G U U U U UreI (urea transporter) U

Circuit Design for U&G Removal

YaaU (guanidine transporter) G pNhaA ureR Turned on by pH sensor Waste Removal

SLIDE 25

25

Phosphate removal

Waste Removal

SLIDE 26

26

Phosphate Regulation Device  Absorb phosphate when: external pH is high and external phosphate level is high  Stop absorbing phosphate when: external pH is low or external phosphate level is low

Waste Removal

SLIDE 27

27

Phosphate Regulation Device

 Ability to absorb external phosphate  Ability to sense Low external phosphate level

Waste Removal

SLIDE 28

28

Absorption of Phosphate

 Ability to absorb phosphate:

Waste Removal

ppk pNhaA pst pTet

Pi Storage Pi Transport (High Affinity and Controllable)

SLIDE 29

29

Sensing of Phosphate

 Ability to sense external low phosphate level:

Waste Removal

SLIDE 30

30

Design of Phosphate Balancing Device Turned on by pH sensor Ter pTet pNhaA pst RBS ppk RBS PPK PST Turned on by low [Pi] tetR Ter pPhoB RBS

TET R

Waste Removal

SLIDE 31

31

Experimental and Modeling Results

31 31

SLIDE 32

32

The pH Sensor

32 32

pH

SLIDE 33

33

High pH sensing

 pNhaA could sense high pH value in environment

33 33

pH

(300uM sodium, 3.5hr) 4.0 8.5 7.0 GFP pH

pNhaA RBS GFP Ter Ter

SLIDE 34

34

Attachment

34

Attach

SLIDE 35

35

Attach

Attachment test

fimH

mpA

lpp gfp

pLac

475/515

10 20 30 40

cell only E.coli+E0840 E.coli+FimH

GFP (475/515 read)

E. coli with GFP detected

by multi-detection microplate reader FimH enhances adherence ability.

E. coli+GFP

+GFP

SLIDE 36

36 36 36

Urea & Guanidine Removal

Waste Removal

SLIDE 37

37 100 200 300 400 500 600 700 800 900 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41

GFP [Urea]

Urea sensing test  The higher urea concentration in environment, the more pUreD activated, and the more GFP expressed.

Waste Removal

ureR pLac GFP pUreD UreR U

SLIDE 38

38 100 200 300 400 500 600 700 800 900 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41

GFP [IPTG] added

U & G transporter test  The more ureI and yaaU activated by pLac, the less urea and guanidine concentration in environment.

38

pLac ureI yaaU Waste Removal [urea] in the environment

SLIDE 39

39

Absorption of Phosphate

Waste Removal

(μM)

removal efficiency = 3.84 ~ 12.8 (pmol/cell)

SLIDE 40

40

Low Phosphate Sensing

40 40

 pPhoB could be activated when phosphate concentration was low in the environment.

Time (hour) GFP

K2HPO4 40uM K2HPO4 200uM K2HPO4 1000uM

Waste Removal

pPhoB RBS GFP Ter Ter

SLIDE 41

Time Regulation

41 41 41

Time

SLIDE 42

Reloxilator Simulation

42 42 0.5 1 1.5 2 2.5 3 1 8 15 22 29 36 43 50 57 64 71 78 85 92 99 106 113 120 127 134 141 148 155 162 169 176 183 190 197

concentration (units) time (units)

Oscillations of CII and HtlB

HtlB CII Oscillator CII Time HtlB CII

SLIDE 43

Reloxilator Assay

Time unit (5 minutes per unit)

Time

SLIDE 44

44 44 44 44

Experimental results

SLIDE 45

45

BacToKidney!!

45 45

pH sensor Attachment Waste removal Time regulation

Future Work:

1. Finish Verifying all functional parts in vitro.
2. Engineer all functional parts into one appropriate

bacterium.

3. Put BacToKidney into animal model.

SLIDE 46

Acknowledgement Special Thanks Funding