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

A Bacteria-based Micro-dialysis Machine For Improving the Quality of Life of Patients Suffering from Chronic Renal Failure 1 1

National Yang Ming University 2

Chronic Renal Failure  Kidneys no longer remove metabolic wastes and maintain balance of body fluid efficiently. http://media.peopo.org/fullscreen.htm?v= Taiwan Broadcasting System (TBS) People Post (PeoPo)  Costing  Inconvenient.  Constrictions on diets.  Injuries of blood vessel, 76 28 fe ultimately causing 54 &auto=true unrecoverable damage . 3 http://www.onemedplace.com/blog/wp- content/uploads/2008/05/soft_case_for_brochure.jpg

Our Solution Traditional BacToKidney Hemodialysis Ability to remove wastes Portable X Time Saving X Self-regulation X 4

Our BacToKidney!! Waste Time pH sensor Attachment removal regulation 5

The pH Sensor pH 6

Why Need pH Sensor? pH  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. 7

How to Sense pH? pH 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 + NhaA H + Na + Derived as pH sensor NhaR nhaA RBS pNhaA 8

Attachment Attach

How to attach? Attach pNhaA ter ter RBS lpp fimH ompA 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

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

How to detach? Attach Time-regulated Protease expression Protease Detach (Time-regulated) ter ter RBS protease Attach Protease cleavage site lpp fimH ter ter ompA RBS pNhaA

Time Regulation Time 13

When to Detach? Time Timer Attachment Detachment Timing time 14

The Timer Time Oscillatory pH System 7 to 8 Input Output Starter AND Stopper time 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. 15

Stopping the Timer Time Accumulated protein amount versus time protein amount Accumulated  Protein produced by Threshold the oscillatory system accumulates. time Output promoter Activity Output promoter activity versus time  After the threshold is Threshold reached, the output promoter of the timer suddenly increases its activity. 16 time

The Oscillatory System Time Oscillatory Cyanoxilator Reloxilator System Input Output AND Starter Stopper Output Oscillatory System Protease 17

The Cyanoxilator (The oscillator with the longer period) Time  Cyanobacterial Oscillator (Cyanoxilator)  A natural oscillator occurring in Cyanobacteria.  Has a period of 14 - 60 hrs. KaiA P P P P Output P P KaiB SasA RpaA Protease P P P P P P P P P KaiC P P Oscillator P 18

The Reloxilator (The oscillator with the shorter period) Time  Relaxation Oscillator (Reloxilator)  A tuneable synchronized oscillator.  Uses parts from: l phage , V. fisheri , and E. coli .  Has a period of around 45 mins. CIIICd HtlB Tuner Output CII CII Protease LuxR LuxI Oscillator Synchronizer 19

Urea & Guanidine Removal Waste Removal 20 20

Why Remove Urea and Guanidine? Waste Removal 21

How to Remove Urea & Guanidine? Waste Removal  Absorption by bacteria (1) Urea transporter (2) Guanidine transporter 22

Absorption of Urea & Guanidine Waste Removal (intestinal lumen) (intestinal cells) (blood) U G G U G U U U U G G U U U U U G G G U U U U G U U Active transporter 23 Passive transporter

Circuit Design for U&G Removal Waste Removal U U Turned on by G pH sensor G U ureR U pNhaA ureR ureR G YaaU U ureR U (guanidine transporter) ureR U G U UreI U (urea transporter) ureI yaaU G pUreD 24

Phosphate removal Waste Removal 25 25

Phosphate Regulation Device Waste Removal  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 26

Phosphate Regulation Device Waste Removal  Ability to absorb external phosphate  Ability to sense Low external phosphate level 27

Absorption of Phosphate Waste Removal  Ability to absorb phosphate: Pi Transport (High Affinity and Controllable) ppk pst pNhaA pTet Pi Storage 28

Sensing of Phosphate Waste Removal  Ability to sense external low phosphate level: 29

Design of Phosphate Balancing Device Waste Removal PST PPK Turned on by pH sensor Ter RBS ppk RBS pst pNhaA pTet Turned on by TET low [Pi] R RBS tetR Ter pPhoB 30

Experimental and Modeling Results 31 31 31

The pH Sensor pH 32 32 32

High pH sensing pH  pNhaA could sense high pH value in environment 8.5 GFP pNhaA RBS Ter Ter GFP 7.0 4.0 pH 33 33 33 (300uM sodium, 3.5hr)

Attachment Attach 34 34 34

Attachment test Attach pLac fimH gfp lpp ompA FimH enhances adherence ability. 40 GFP (475/515 read) 30 475/515 20 E. coli with GFP detected 10 by multi-detection microplate reader 0 +GFP cell only E.coli+FimH E. coli+GFP 35 E.coli+E0840

Urea & Guanidine Removal Waste Removal 36 36 36

Urea sensing test Waste Removal  The higher urea concentration in environment, the more pUreD activated, and the more GFP expressed. 900 800 700 GFP 600 500 400 U 300 UreR 200 ureR GFP 100 pLac pUreD 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 37 [Urea]

U & G transporter test Waste Removal  The more ureI and yaaU activated by pLac , the less urea and guanidine concentration in environment. pLac 900 [urea] in the environment ureI yaaU 800 700 GFP 600 500 400 300 200 100 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 38 38 [IPTG] added

Absorption of Phosphate Waste Removal ( μ M) removal efficiency = 3.84 ~ 12.8 (pmol/cell) 39

Low Phosphate Sensing Waste Removal  pPhoB could be activated when phosphate concentration was low in the environment. GFP pPhoB RBS Ter Ter GFP K 2 HPO 4 40uM K 2 HPO 4 200uM K 2 HPO 4 1000uM Time (hour) 40 40 40

Time Regulation Time 41 41 41

Reloxilator Simulation Time Oscillations of CII and HtlB HtlB 3 CII CII 2.5 concentration (units) Oscillator 2 1.5 HtlB 1 0.5 CII 0 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 42 42 time (units)

Reloxilator Assay Time Time unit (5 minutes per unit)

Experimental results 44 44 44 44

BacToKidney!! Waste Time pH sensor Attachment Future Work: removal regulation 1. Finish Verifying all functional parts in vitro . 2. Engineer all functional parts into one appropriate bacterium. 3. Put BacToKidney into animal model. 45 45 45

Acknowledgement Special Thanks Funding

BacToKidney!! THANKS FOR LISTENING  NYMU-Taipei iGEM 2008 offical wikipage http://2008.igem.org/Team:NYMU-Taipei 47