1. Synthetic Gene Networks y
That CountThat Count
Ari E. Friedland, Timothy K. Lu, Xiao Wang, David Shi, GeorgeAri E. Friedland, Timothy K. Lu, Xiao Wang, David Shi, George
Church, James J. Collins.
( )SCIENCE 324 (2009) pp. 1199‐1202
A IgolkinaA. Igolkina
1

2. R i + M ti tiReview + Motivation
• What do we need for a counting? g
Memory + Mechanism
• Natural memory:
– DNA sequence.
– Level of protein concentration. (Temporary, but could be not very
short in time)
• There are a lot of stable mechanisms in a cell.
• Steps to biocomputers:
– Whole‐cell biocomputing. TRENDS in Biotechnology 19 (2001).
L i l ti AND d OR• Logical operation AND and OR.
– Programmable single‐cell mammalian biocomputers. Nature 487
(2012).( )
• An idea of digital computations with NOT, AND, NAND and N‐IMPLY expression logic
in single mammalian cells.
h ’ ll ? h !• Why can’t cells count? They can!
2

3. R i + M ti tiReview + Motivation
• What do we need for a counting? g
Memory + Mechanism
• Natural memory:
– DNA sequence.
– Level of protein concentration. (Temporary, but could be not very
short in time)
• There are a lot of stable mechanisms in a cell.
• Steps to biocomputers:
– Whole‐cell biocomputing. TRENDS in Biotechnology 19 (2001).
L i l ti AND d OR• Logical operation AND and OR.
– Programmable single‐cell mammalian biocomputers. Nature 487
(2012).( )
• An idea of digital computations with NOT, AND, NAND and N‐IMPLY expression logic
in single mammalian cells.
h ’ ll ? h !• Why can’t cells count? They can!
2

4. R i + M ti tiReview + Motivation
• What do we need for a counting? g
Memory + Mechanism
• Natural memory:
– DNA sequence.
– Level of protein concentration. (Temporary, but could be not very
short in time)
• There are a lot of stable mechanisms in a cell.
• Steps to biocomputers:
– Whole‐cell biocomputing. TRENDS in Biotechnology 19 (2001).
L i l ti AND d OR• Logical operation AND and OR.
– Programmable single‐cell mammalian biocomputers. Nature 487
(2012).( )
• An idea of digital computations with NOT, AND, NAND and N‐IMPLY expression logic
in single mammalian cells.
h ’ ll ? h !• Why can’t cells count? They can!
2

5. R i + M ti tiReview + Motivation
• What do we need for a counting? g
Memory + Mechanism
• Natural memory:
– DNA sequence.
– Level of protein concentration. (Temporary, but could be not very
short in time)
• There are a lot of stable mechanisms in a cell.
• Steps to biocomputers:
– Whole‐cell biocomputing. TRENDS in Biotechnology 19 (2001).
L i l ti AND d OR• Logical operation AND and OR.
– Programmable single‐cell mammalian biocomputers. Nature 487
(2012).( )
• An idea of digital computations with NOT, AND, NAND and N‐IMPLY expression logic
in single mammalian cells.
h ’ ll ? h !• Why can’t cells count? They can!
2

6. C ti h iCounting mechanism
I’m a cell I’ve got a memoryI m a cell. I ve got a memory.
Now I remember number 0!
3

7. C ti h iCounting mechanism
Add one, please.
00
3

8. C ti h iCounting mechanism
Ok. Good.
11
3

9. C ti h iCounting mechanism
Add one, please.
11
3

10. C ti h iCounting mechanism
Ok. Good.
22
“Memory” : concentration of some proteinMemory : concentration of some protein
Regulation : extrinsical chemical stimulius
3

11. Model 1: Riboregulated transcriptional g p
cascade(RTC), two‐counter
• The cis‐repressor sequence (cr) is complimentary with ribosome binding site (RBS).
• A stem‐loop in a secondary structure of RNA prevents binding of ribosome (30S).
• A short noncoding taRNA binds to the cr; the stem‐loop can’t form; translation is allowed.
• Only T7 polymerase, which is coded by T7RNAP, can bind to promoter PT7.O y po y e ase, c s coded by , ca b d to p o ote .
• A transcription of taRNA depends on concentration pulses of arabinose.
4

12. Model 1: Riboregulated transcriptional g p
cascade(RTC), two‐counter
5

13. Model 1: Riboregulated transcriptional g p
cascade(RTC), three‐counter
6

14. E i t l lt f RTC tExperimental results of RTC counters
Two counter Three counterTwo‐counter Three‐counter
Shaded areas represent arabinose pulse
l l d h fl l• Experimental results demonstrate that fluorescence increases only
when all two (or three) arabinose pulses are delivered.
• Based on this results, Ari E. Friedland at el. constructed and analyzed aBased on this results, Ari E. Friedland at el. constructed and analyzed a
mathematical model.
7

15. P di ti f th th ti l d lPrediction of the mathematical model
Two counter Maximum of the predicted concentration.Two‐counter
Three‐counter: searching of
Maximum of the predicted concentration.
Three counter: searching of
optimal combination
(interval‐length).
Points: the differencePoints: the difference
between model and
experiment.
Three‐counter
The absolute difference in
fluorescence after three
pulses and two
8

16. Model 2: Single Invertase Memory g y
Module(SIMM), three‐counter
DNA – vector
• After the first arabinose pulse only Flpe protein can express.
• Flpe is a site‐specific recombinase. (Site FRT)
9

17. Model 2: Single Invertase Memory g y
Module(SIMM), three‐counter
10

18. Model 2: Single Invertase Memory g y
Module(SIMM), three‐counter
• After the second arabinose pulse only Cre protein can express.
• Cre is a site‐specific recombinase. (Site lox)
11

19. Model 2: Single Invertase Memory g y
Module(SIMM), three‐counter
Counting cascade.
Result: GFP expresses after 3Result: GFP expresses after 3
Ara pulses.
The same cascade was built forThe same cascade was built for
two‐counter
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20. Experimental results and Modelling of
SIMM counters
Fluorescence in SIMM three‐counter aftres 3 pulses.
GFP fluorescence ratios between the single‐inducer
DIC h d h l fDIC three‐counter exposed to three pulses of
arabinose (N) versus two pulses of arabinose (N – 1)
with varying arabinose pulse lengths and intervals;
experimental results are represented by black dots.
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21. C l i + F tConclusion + Future
• Imitation of computer ticks.
• Temporary effect.
• Unfortunately cell mechanisms have got• Unfortunately, cell mechanisms have got
mistakes and this counters too.
• There is a dubious future for biocompurets
which are based on this counterswhich are based on this counters.
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22. Thank for your attention!f y