4-State Device

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-
= Organisation =
+
= Purpose =
 +
The 4-State Device (4SD) uses two inputs to sequentially switch through its four states. The four states are used to keep track of changes of the inputs. One state is connected to the output.
-
=== Group Members ===
+
= Design =
-
*[[Alexander Roth]](coordinator),  [[User:realUACM|Urs A. Müller]],  [[Simon Barkow]],  [[User:Tamara|Tamara Ulrich]],  [[Robin Künzler]], Herve, Dominic, Christophe ...
+
-
=== [[NOR Meetings]] ===
+
== Suitable Strategies ==
 +
For realizing the design we need one repressor for each state. For this purpose there are several possibilities that can be considered:
-
= Description of the NOR module =
+
* Silencing approach (antisense RNAs, short hairpin RNAs (shRNAs)) to reduce the pool of translatable mRNAs
-
This module behaves like a NOR gate. It has two inputs and one output. The output is high only when neither input 1 nor input 2 is high.
+
-
== NOR Gate ==
+
* Existing Repressor-Operator (for example TetR-TetO)
-
In the figure below is a parts-view of the NOR gate module with PoPS interfaces (i/o).
+
-
              ---------------------------------
+
* Designing new repressors (for example Zinc Finger Proteins)
-
              |  ------------\                |
+
-
PoPS_in1 ---->|  | Repressor1 | --------      |
+
-
              |  ------------/          |    |
+
-
              |                        =    |
+
-
              |                      --------|------> PoPS_out
+
-
              |                        =    |
+
-
              |  ------------\          |    |
+
-
PoPS_in2 ---->|  | Repressor2 | --------      |
+
-
              |  ------------/                |
+
-
              ---------------------------------
+
-
== Interconnected NOR module ==
+
== Selected System ==
 +
Considering the modularity as the first criterion for the selection of the system, we considered that the Zinc Finger Protein (ZFP) approach would be optimal. Indeed, the operators could be as short as 9 nucleotide long and the specificity can be easily increased by either increasing the number of fingers in the ZFP or by coupling ZFP together via Leucine Zippers.
 +
The recognition sequence can then be scaled up to 18 nucleotides or even further.
 +
Known repressors such as tetR remain a possibility for our system because of the tunability that this repressor-operator can offer (via tetracycline for the TetR-TetO couple).
 +
The silencing approach has been dropped because of the lack of modularity and the difficulty to achieve full repression via silencing approaches in bacteria.
 +
The selected approach (ZFP) allowed us to put the binding sites for the repressors directly after start of transcription and before the ribosome binding site to prevent RNA polymerase to transcribe the gene. This design allows us to put any promoter in front of the binding site. This option gives a very high degree of modularity.
-
= Design =
+
When we decide to scale up the number of counters in order to count n^2, we need to design 4 new repressors for each 4 state device. With the Zinc Finger Protein approach we can easily design new sequence specific repressors based on the ZFP - DNA recognition motif database available.
-
== Repressors ==
+
= Actual Implementation =
-
The Interconnected NOR module consists of four repressors. We considered several alternatives but finally decided to put all out bets on a approach based on zinc finger proteins.  
+
The central part of the counter is the 4SD. When input changes the next state is reached and the repressor is expressed. Every Repressor represses all other states except for the next. For example R1 represses R3 and R4, but not R2. This forces a progression of states R1 to R4 during the cycles of the input.
-
RNA polymerase...
+
In the figure below is a parts-view of the device with PoPS interfaces (i/o).
-
=== Zinc Finger Proteins ===
+
==== Basic Functionality ====
-
A Zinc Finger (ZF) [http://en.wikipedia.org/wiki/Zinc_finger] is a protein domain [http://en.wikipedia.org/wiki/Protein_domain] that binds to three base pairs of double stranded DNA. A Zinc Finger Protein (ZFP) consists of one or several zinc finger domains.  Many protein-DNA interaction for ZF domains and triplet of base pairs have been described, therefore making it possible to to construct artificial transcription factors by combining ZF domains in a modular fashion. The idea is to use a ZFP as a repressor by putting a binding site for a ZFP upstream of the coding region and thereby preventing RNA polymerase to transcribe the gene.
+
The 4-State Device uses two inputs to sequentially switch through its four states. Note that this behavior can be observed abstractly in the concept section.  
-
We use several different designs of ZFPs.  
+
To achieve such behavior in our system, we use four interconnected gates. In the following, we call them ''NOR gates''. Each of our NOR gates has three inputs. All of them have to be low for the output to be high. In boolean logic, this reads as out=(NOT_in_1 ^ NOT_in_2 ^ ... ^ NOT_in_n)  <b>(i can't figure out how this f*****'  math mode works! Can someone more savvy put this in math mode?, jonas</b>
-
- Three ZF domains to recognize nine base pairs.
+
===== Electrical NOR gate =====
 +
A NOR gate is a very common part in electrical engineering.
-
- Two ZF domains and a leucine zipper domain. The leucine zipper will be cause the protein to home-dimerize and hence it will recognize 12 base pairs.
+
[[Image:NORcircuit.gif]]
-
- Six ZF domains to recognize twelve base pairs.
+
===== Biological NOR gate =====
 +
Biologically, a NOR gate can be implemented through a promoter with high basal activity that is repressed by three effectors. If there is at least one of the three repressors, transcription is inhibited.
 +
Each NOR gate expresses a repressor protein. (Note that such a biological NOR gate has no POPs interfaces)
-
- Three ZF domains fused to negative transcription factors. (e.g. NTD and ERD)
+
[[Image:NOR.gif]]
 +
===== 4-State Device circuit diagram =====
-
=== Zinc Finger Protein Binding Sites ===
+
[[Image:4SDcircuit.gif]]
-
Since we are going to synthesize parts of our module, we have the possibility to design our own ZFP binding sites, as well as our ZFPs.
+
-
We have two alternatives for placing the binding sites.  
+
-
1. Binding sites in the promoter. This would prevent the polymerase from binding to the promotor. Although this might be most likely to work, we have chosen to not pursue this alternative, while we are quite intrigued by the possibility of a roadblock.
+
===== 4-State Device parts diagram =====
-
2. Binding sites directly after the start of transcription and before the ribosome binding site. This alternative is attractive, since it would allow for a high degree of modularity. In theory the ZFP would act as an extra "roadblock-operator" and any promotor could be used in front of the protein.
+
[[Image:4StateDevice.png]]
-
The ZFP roadblock operator regions (from now on refered to as: operators) consists of binding motifs for two repressors. The two bindning motifs are usually spaced with 5 base pairs (gcgcg). Some data of the binding strength were available and we have chosen operators with the affinity estimated to K_d = 3-40 nM.
+
In the design of the Event Processing Device, input 1 and input 2 have opposite activity, meaning that either R1/R3 or R2/R4 is active. Furthermore, since R1 and R3 (respectively R2 and R4) are repressing each other, only one of the two is active. Therefore, in a stable situation, only 1 of the 4 repressor proteins is expressed.
-
{| border="1" cellpadding="2"
+
Let us assume that R1 is being expressed. Input 2 must then be low, and therefore input 1 high. This situation is stable and remains until there is a change in the inputs. Now, if input 1 decreases, and input 2 increases, the expression of R1 will come to a halt. Since input 1 is now low, either R2 or R4 will be expressed. At this stage, R1 is still present in relatively high concentration and by repressing R4, it tips the balance in favor of R2, leading to a new stable state in which only R2 is expressed.
-
! Repressor !! Binding motif !! K_d (nM) !! freq. in genome !! freq. hamming dist=1  
+
-
|-
+
-
| R1       || ggaggggac    || 4        || 1               || 132
+
-
|-
+
-
| R2       || ggaggcggg    || 30      || 5              || 396
+
-
|-
+
-
| R3        || gggggcgag    || 3        || 8              || 303
+
-
|-
+
-
| R4       || ggggccgga    || 45      || 23              || 433
+
-
|-
+
-
| R1 lz    || gtccccggggac  || N/A      || 0              || 2
+
-
|-
+
-
| R2 lz    || ctcgccggcgag  || N/A      || 0              || 7
+
-
|-
+
-
| R3 lz    || cccgccggcggg  || N/A      || 0              || 19
+
-
|-
+
-
| R4 lz    || tccggcgccgga  || N/A      || 0              || 27
+
-
|}
+
-
We need to have 4 operators = 4 permutations of ZFP.
+
Note that electrical engineers call such a device a "J-K flip flop". It can also be seen as a combination of two toggle switches ([[Atkinson03]]), each being able to store one bit.
-
The operator regions (i.e. the "roadblocks" that will prevent of the RNApolymerase to transcribe the gene) form a BioBrick that should be inserted between the promoter region and the ribosome binding site in order to keep the design modular.  
+
-
== Zinc Finger Tester ==
+
=== Biological Details ===
-
To test whether our assumptions about using multiple zinc finger proteins (ZFP) as repressors (i.e. roadblocks) will actually work, we will build a tester/debugging device in parallel with the counter.
+
-
== Interconnected NOR System ==
+
In the design of the repressor protein we use the following domains:
-
The NOR system consists of 4 proteins with 4 operator regions. It has an interface boundary with input module (Pr and Prm). Repressor R3 is connected to a reporter to be able to count to modulo two.
+
 +
- ''Zinc Finger (ZF)''
 +
DNA binding domain
 +
- ''NTD''
 +
The N-terminal polymerase domain from NusA. This protein has a RNA polymerase halting properties.
-
=== Repression ===
+
- ''ERD''
-
Note that in both cases, it is still under discussion as whether the repression domain should really be included or not. If it turns out that repression has been used in all the literature we (well, actually Hervé) can find, then we would be better off to play safe and include them:
+
ERF repressor domain. a repressor domain in eukaryotes.
-
* Beerli PNAS 1998: they fused ZF to KRAB repressor and it has a stronger effect compare to ZF alone (see p14632 graph A)
+
-
* Beerli Nat Biotech review feb2002 : if you read the complete paragraph on gene repression (p 132), it gives strong evidence that we should fuse the ZF to a repressor (apparently at the N-Term of the ZF) (either KRAB or SID repressor). It is stated that polymerase blockade through ZF only is not very efficient.
+
-
However, the discussion is ongoing: As Alex pointed out we can not be sure that the 45 amino acid long KRAB domain will work, if it don't exists in prokaryotes. KRAB domains have a very specific interaction with a co-repressor molecule. It might be better to find something already known to work in bacteria or skip the repressor approach altogether.
+
-
An alternative could be a negative transcription domain fused to the ZFP.
+
-
=== Degradation ===
+
- ''LZ''
-
We considered adding a variant with degradation tag, since the system might be slow otherwise. However, due to the fact that we have limited resources - in terms of money, manpower, and time - and the fact that the degradation is decoupled and it will be clearly visible if the system turns out to be too slow, we decided against it for now.
+
Leucine zipper. For dimerization.
-
=== Additional Comments ===
+
If it turns out that repression has been used in all the literature we can find, then we would be better off to play safe and include them:
-
We changed our strategy a couple of times in heated debates. There is quite obviously a trade-off between probability of success through redundancy, i.e. trying multiple approaches in parallel, and limited resources, i.e. financial, manpower, and time. Thus we decided against certain additional variants and cloning steps since we simply won't have the time for it.
+
-
Another important aspect is the overall goal of keeping the design modular - one of the most important aspects of the this contest.
+
-
The modification of the promoter regions of the lambda-system, i.e. Pr and Prm, although a valid approach will probably be dropped, since it is too much work and cost and no longer modular (as opposed to having the operators after the promoter).
+
-
There has been some discussion about having the operator around 0 (-9/+1)
+
* Beerli PNAS 1998: they fused ZF to KRAB repressor and it has a stronger effect compare to ZF alone (see p14632 graph A)
 +
* Beerli Nat Biotech review feb2002 : if you read the complete paragraph on gene repression (p 132), it gives strong evidence that we should fuse the ZF to a repressor (apparently at the N-Term of the ZF)  
 +
It is stated that polymerase blockade through ZF only is not very efficient.
 +
We can not be sure that the 45 amino acid long KRAB domain will work, if it doesn't exist in prokaryotes. KRAB domains have a very specific interaction with a co-repressor molecule. It might be better to find something already known to work in bacteria or skip the repressor approach altogether.
-
= Synthesis and Assembly =
 
 +
The different designs of repressors
{| border="1" cellpadding="2"
{| border="1" cellpadding="2"
-
|+Sequences to Order
+
|+ Synthesized Sequences
-
! Seq nickname  !! Length !! Cost    !! SacI !! KpnI !! NheI !! EcoRI !! XbaI  !! SpeI  !! PstI
+
! BioBrick Part !! Number of ZF domains !! LZ domain !! TF domain
|-
|-
-
| J05211+J05100 || 460    || $736.00  || 1   || 74  || 455  || 7,80  || 22,95 || 54,435 || 68,449
+
| BBa_J05101 || 3  || -    ||    -
|-
|-
-
| J05212+J05102 || 460    || $736.00  || 1    || 74  || 455  || 7,80  || 22,95 || 54,435 || 68,449
+
| BBa_J05108 || 3* || CREBH || -
|-
|-
-
| J05213+J05101 || 460    || $736.00 || 1    || 74  || 455  || 7,80  || 22,95 || 54,435 || 68,449
+
| BBa_J05109 || 3* || ATF6 || -
|-
|-
-
| J05214+J05103 || 460    || $736.00 || 1    || 74  || 455  || 7,80  || 22,95 || 54,435 || 68,449
+
| BBa_J05110 || 3* || YAP7 || -
|-
|-
-
| J05218+J05108 || 515    || $824.00 || 1    || 81  || 510  || 7,87  || 22,102|| 61,490 || 75,504
+
| BBa_J05111 || 3* || cMaf || -
|-
|-
-
| J05109        || 414    || $662.40  || 1    || 409 ||-    || 7    || 22    || 389    || 403
+
| BBa_J05112 || 3 || -    || ERD
|-
|-
-
| J05222+J05115 || 676    || $1081.60 || 1    || 87  ||671  || 7,93 || 22,108|| 67,651 || 81,665
+
| BBa_J05113 || 3* || ATF6 || KRAB
|-
|-
-
| J05221+J05114 || 728    || $1164.80 || 1    || 133 || 723  || 7,139 || 22,154|| 113,703|| 127,717
+
| BBa_J05114 || 3* || ATF6 || NTD
|-
|-
-
| J05112        || 524    || $838.40  || 1    || 519 || -   || 7     || 7    || 499    || 513
+
| BBa_J05115 || 6 || -    || -
 +
|}
 +
 
 +
 
 +
 
 +
==== BioBrick parts ====
 +
 
 +
{| border="1" cellpadding="2"
 +
|+ BBa_J05040 4-State Device
 +
! State !! Repressor  !! Regulator !! Comments
|-
|-
-
| J05215+J05108  || 519 || $830.40 || || || || || || ||
+
| R1 || BBa_J05311 || BBa_J05215 ||  
|-
|-
-
| J05216+J05109  || 498 || $796.80 || || || || || || ||
+
| R3 || BBa_J05312 || BBa_J05216 ||  
|-
|-
-
| J05217+J05110  || 525 || $840.00 || || || || || || ||
+
| R2 || BBa_J05313 || BBa_J05217 ||  
|-
|-
-
| J05218+J05111  || 516 || $825.60 || || || || || || ||
+
| R4 || BBa_J05314 || BBa_J05218 ||  
|}
|}
-
== ZFP Tester ==
 
-
* Cut each system into BioBricks Pieces using different restriction enzymes.
 
-
* Assemble in parallel for each genes 1-4 in two variants: promoter+operator, rbs+gene
 
-
* Assemble the two constructs
 
-
* ...
 
-
[[Image:TesterParts.gif]]
 
-
=== Tests ===
 
{| border="1" cellpadding="2"
{| border="1" cellpadding="2"
-
|+Repression Tests
+
|+ Synthesized Sequences
-
! Repressor !! Operator !! Comments
+
! Nickname              !! Length !! SacI !! KpnI !! NheI !! EcoRI !! XbaI    !! SpeI    !! PstI
|-
|-
-
| J05100    || J05212   || ...
+
| BBa_J05215+BBa_J05108 || 603   || 1  ||  91  ||  598 || 7, 97  || 22, 112 || 71, 578 || 85, 592
|-
|-
-
| J05100     || J05212   || ...
+
| BBa_J05216+BBa_J05109 ||  582  ||  1  ||  91  ||  577 || 7, 97  || 22, 112 || 71, 557 || 85, 571
 +
|-
 +
| BBa_J05217+BBa_J05110 ||  609  ||  1  ||  91  ||  604 || 7, 97  || 22, 112 || 71, 584 || 85, 598
 +
|-
 +
| BBa_J05218+BBa_J05111 ||  600  ||  1  ||  91  ||  595 || 7, 97  || 22, 112 || 71, 575 || 85, 589
 +
|-
 +
| BBa_J05221+BBa_J05101 ||  498  ||  1  ||  112 ||  493 || 7, 118 || 22, 133 || 92, 473 || 106, 487
 +
|-
 +
| BBa_J05114            ||  854  ||  1  ||  849 ||  -  ||    7  || 22      || 829     || 843
 +
|-
 +
| BBa_J05112            ||  524   || 1  ||  519 ||  -  ||    7  || 22      || 499    || 513
 +
|-
 +
| BBa_J05222+BBa_J05115 ||  676  ||  1  ||  87  ||  671 || 7, 93  || 22, 108 || 67, 651 || 81, 665
|}
|}
-
(complete this...)
 
-
== Interconnected NOR device ==
 
-
[[Image:CounterParts.gif]]
+
The provider of the synthesized DNA used for the zinc fingerers has a lower cost limit of $500. To reduce cost per base pair for short sequences we put the short sequences together with a long sequence with restriction sites between them. the trick we use to reduce cost, by having an extra step of cloning.
-
=References=
+
binding of ZFP. describe choice of binding sites. Describe the function of RNA pol II. emphasize: modularity!
-
[[Kim & Wang]] (??)
+
Another important aspect is the overall goal of keeping the design modular - one of the most important aspects of this contest.
-
[[Park et al.]] 2005 (Activation of transcription)
 
-
[[Chou et al.]] 1998
 
-
[[Mani05]]
+
We have two alternatives for placing the binding sites.
 +
 +
1. Binding sites in the promoter. This would prevent the polymerase from binding to the promotor. Although this might be most likely to work, we have chosen to not pursue this alternative, while we are quite intrigued by the possibility of a roadblock. The operator regions (i.e. the "roadblocks" that will prevent of the RNApolymerase to transcribe the gene) form a BioBrick that should be inserted between the promoter region and the ribosome binding site in order to keep the design modular.
-
[[Beerli98]]
+
2. Binding sites directly after the start of transcription and before the ribosome binding site. This alternative is attractive, since it would allow for a high degree of modularity. In theory the ZFP would act as an extra "roadblock-operator" and any promotor could be used in front of the protein.
-
[[Beerli00]] (Linker)
+
The ZFP roadblock operator regions (from now on referred to as: operators) consists of binding motifs for two repressors. The two binding motifs are usually spaced with 5 base pairs (gcgcg). Some data of the binding strength were available and we have chosen operators with the affinity estimated to K_d = 3-40 nM.
-
[[Beerli02]]
+
{| border="1" cellpadding="2"
 +
!    Part  !!  Binding Site Seq !! est Kd [nM] !! # exact matches !! Hamming Dist 1 !! Hamming Dist 2 !! Hamming Dist 3
 +
|-
 +
| BBa_J05100 || ggaggggac          || 4  || 5  || 267 || 6196  || 65733
 +
|-
 +
| BBa_J05102 || ggaggcggg          || 30  || 17 || 799 || 12589 || 95996
 +
|-
 +
| BBa_J05101 || gggggcgag          || 3  || 15 || 634 || 10173 || 82956
 +
|-
 +
| BBa_J05103 || ggggccgga          || 45  || 44 || 848 || 11317 || 91422
 +
|-
 +
| BBa_J05108 || gtcccctccggaggggac || N/A || 0  || 0  || 0    || 1
 +
|-
 +
| BBa_J05109 || ctcgcccccgggggcgag || N/A || 0  || 0  || 0    || 2
 +
|-
 +
| BBa_J05110 || cccgcctccggaggcggg || N/A || 0  || 0  || 0    || 4
 +
|-
 +
| BBa_J05111 || tccggccccggggccgga || N/A || 0  || 0  || 0    || 6
 +
|-
 +
| BBa_J05115 || gggggcgaggggggcgag || N/A || 0  || 0  || 0    || 3
 +
|}
-
[[Isalan01]]
 
-
[[Greisman97]]
 
-
[[Segal03]]
+
Degradation rates????
-
[[Segal99]] ("GNN-paper")
+
Tests: design all tests.
 +
To test whether our assumptions about using multiple zinc finger proteins (ZFP) as repressors (i.e. roadblocks) will actually work, we will build a tester/debugging device in parallel with the counter.
-
[[Dreier01]] ("ANN-paper")
 
-
[[Dreier05]] ("CNN-paper")
+
{| border="1" cellpadding="2"
 +
|+ BBa_J05000 Zinc Finger Protein Tester
 +
! Repressor  !! Regulator !! Comments
 +
|-
 +
| BBa_J05112 R3-e2+ERD || Regulator for R3-ATF6 ||
 +
|-
 +
| BBa_J05113 R3-ATF6+ NTD || BBa_J05221 Triple Binding Site for R3-ATF6 ||
 +
|-
 +
| BBa_J05114 R3-ATF6+NTD || Regulator for R3-ATF6 ||
 +
|-
 +
| BBa_J05101 R3-e2 || Regulator for R3-ATF6 ||
 +
|-
 +
| BBa_J05115 ZF-2*e2 || ZF-2*e2 Binding Site ||
 +
|}
-
[[Yang95]] (Kinetics!!)
 
-
[[Klug05]] (Minireview)
+
assembly diagram of test.
 +
[[Image:TesterParts.gif]]
-
[[Newman03]] ("Leucine Zippers")
+
 
 +
4SD system
 +
The 4SD system consists of 4 proteins with 4 operator regions. It has an interface boundary with input module (Pr and Prm). Repressor R3 is connected to a reporter to be able to count to modulo two.
 +
assembly diagram of device.
 +
[[Image:CounterParts.gif]]

Latest revision as of 19:58, 5 November 2005

Back to the ETH Zurich main page.

Contents

Purpose

The 4-State Device (4SD) uses two inputs to sequentially switch through its four states. The four states are used to keep track of changes of the inputs. One state is connected to the output.

Design

Suitable Strategies

For realizing the design we need one repressor for each state. For this purpose there are several possibilities that can be considered:

  • Silencing approach (antisense RNAs, short hairpin RNAs (shRNAs)) to reduce the pool of translatable mRNAs
  • Existing Repressor-Operator (for example TetR-TetO)
  • Designing new repressors (for example Zinc Finger Proteins)

Selected System

Considering the modularity as the first criterion for the selection of the system, we considered that the Zinc Finger Protein (ZFP) approach would be optimal. Indeed, the operators could be as short as 9 nucleotide long and the specificity can be easily increased by either increasing the number of fingers in the ZFP or by coupling ZFP together via Leucine Zippers. The recognition sequence can then be scaled up to 18 nucleotides or even further.

Known repressors such as tetR remain a possibility for our system because of the tunability that this repressor-operator can offer (via tetracycline for the TetR-TetO couple).

The silencing approach has been dropped because of the lack of modularity and the difficulty to achieve full repression via silencing approaches in bacteria.

The selected approach (ZFP) allowed us to put the binding sites for the repressors directly after start of transcription and before the ribosome binding site to prevent RNA polymerase to transcribe the gene. This design allows us to put any promoter in front of the binding site. This option gives a very high degree of modularity.

When we decide to scale up the number of counters in order to count n^2, we need to design 4 new repressors for each 4 state device. With the Zinc Finger Protein approach we can easily design new sequence specific repressors based on the ZFP - DNA recognition motif database available.

Actual Implementation

The central part of the counter is the 4SD. When input changes the next state is reached and the repressor is expressed. Every Repressor represses all other states except for the next. For example R1 represses R3 and R4, but not R2. This forces a progression of states R1 to R4 during the cycles of the input.

In the figure below is a parts-view of the device with PoPS interfaces (i/o).

Basic Functionality

The 4-State Device uses two inputs to sequentially switch through its four states. Note that this behavior can be observed abstractly in the concept section.

To achieve such behavior in our system, we use four interconnected gates. In the following, we call them NOR gates. Each of our NOR gates has three inputs. All of them have to be low for the output to be high. In boolean logic, this reads as out=(NOT_in_1 ^ NOT_in_2 ^ ... ^ NOT_in_n) (i can't figure out how this f*****' math mode works! Can someone more savvy put this in math mode?, jonas

Electrical NOR gate

A NOR gate is a very common part in electrical engineering.

NORcircuit.gif

Biological NOR gate

Biologically, a NOR gate can be implemented through a promoter with high basal activity that is repressed by three effectors. If there is at least one of the three repressors, transcription is inhibited. Each NOR gate expresses a repressor protein. (Note that such a biological NOR gate has no POPs interfaces)

NOR.gif

4-State Device circuit diagram

4SDcircuit.gif

4-State Device parts diagram

4StateDevice.png

In the design of the Event Processing Device, input 1 and input 2 have opposite activity, meaning that either R1/R3 or R2/R4 is active. Furthermore, since R1 and R3 (respectively R2 and R4) are repressing each other, only one of the two is active. Therefore, in a stable situation, only 1 of the 4 repressor proteins is expressed.

Let us assume that R1 is being expressed. Input 2 must then be low, and therefore input 1 high. This situation is stable and remains until there is a change in the inputs. Now, if input 1 decreases, and input 2 increases, the expression of R1 will come to a halt. Since input 1 is now low, either R2 or R4 will be expressed. At this stage, R1 is still present in relatively high concentration and by repressing R4, it tips the balance in favor of R2, leading to a new stable state in which only R2 is expressed.

Note that electrical engineers call such a device a "J-K flip flop". It can also be seen as a combination of two toggle switches (Atkinson03), each being able to store one bit.

Biological Details

In the design of the repressor protein we use the following domains:

- Zinc Finger (ZF) DNA binding domain

- NTD The N-terminal polymerase domain from NusA. This protein has a RNA polymerase halting properties.

- ERD ERF repressor domain. a repressor domain in eukaryotes.

- LZ Leucine zipper. For dimerization.

If it turns out that repression has been used in all the literature we can find, then we would be better off to play safe and include them:

  • Beerli PNAS 1998: they fused ZF to KRAB repressor and it has a stronger effect compare to ZF alone (see p14632 graph A)
  • Beerli Nat Biotech review feb2002 : if you read the complete paragraph on gene repression (p 132), it gives strong evidence that we should fuse the ZF to a repressor (apparently at the N-Term of the ZF)

It is stated that polymerase blockade through ZF only is not very efficient. We can not be sure that the 45 amino acid long KRAB domain will work, if it doesn't exist in prokaryotes. KRAB domains have a very specific interaction with a co-repressor molecule. It might be better to find something already known to work in bacteria or skip the repressor approach altogether.


The different designs of repressors

Synthesized Sequences
BioBrick Part Number of ZF domains LZ domain TF domain
BBa_J05101 3 - -
BBa_J05108 3* CREBH -
BBa_J05109 3* ATF6 -
BBa_J05110 3* YAP7 -
BBa_J05111 3* cMaf -
BBa_J05112 3 - ERD
BBa_J05113 3* ATF6 KRAB
BBa_J05114 3* ATF6 NTD
BBa_J05115 6 - -


BioBrick parts

BBa_J05040 4-State Device
State Repressor Regulator Comments
R1 BBa_J05311 BBa_J05215
R3 BBa_J05312 BBa_J05216
R2 BBa_J05313 BBa_J05217
R4 BBa_J05314 BBa_J05218


Synthesized Sequences
Nickname Length SacI KpnI NheI EcoRI XbaI SpeI PstI
BBa_J05215+BBa_J05108 603 1 91 598 7, 97 22, 112 71, 578 85, 592
BBa_J05216+BBa_J05109 582 1 91 577 7, 97 22, 112 71, 557 85, 571
BBa_J05217+BBa_J05110 609 1 91 604 7, 97 22, 112 71, 584 85, 598
BBa_J05218+BBa_J05111 600 1 91 595 7, 97 22, 112 71, 575 85, 589
BBa_J05221+BBa_J05101 498 1 112 493 7, 118 22, 133 92, 473 106, 487
BBa_J05114 854 1 849 - 7 22 829 843
BBa_J05112 524 1 519 - 7 22 499 513
BBa_J05222+BBa_J05115 676 1 87 671 7, 93 22, 108 67, 651 81, 665


The provider of the synthesized DNA used for the zinc fingerers has a lower cost limit of $500. To reduce cost per base pair for short sequences we put the short sequences together with a long sequence with restriction sites between them. the trick we use to reduce cost, by having an extra step of cloning.

binding of ZFP. describe choice of binding sites. Describe the function of RNA pol II. emphasize: modularity! Another important aspect is the overall goal of keeping the design modular - one of the most important aspects of this contest.


We have two alternatives for placing the binding sites.

1. Binding sites in the promoter. This would prevent the polymerase from binding to the promotor. Although this might be most likely to work, we have chosen to not pursue this alternative, while we are quite intrigued by the possibility of a roadblock. The operator regions (i.e. the "roadblocks" that will prevent of the RNApolymerase to transcribe the gene) form a BioBrick that should be inserted between the promoter region and the ribosome binding site in order to keep the design modular.

2. Binding sites directly after the start of transcription and before the ribosome binding site. This alternative is attractive, since it would allow for a high degree of modularity. In theory the ZFP would act as an extra "roadblock-operator" and any promotor could be used in front of the protein.

The ZFP roadblock operator regions (from now on referred to as: operators) consists of binding motifs for two repressors. The two binding motifs are usually spaced with 5 base pairs (gcgcg). Some data of the binding strength were available and we have chosen operators with the affinity estimated to K_d = 3-40 nM.

Part Binding Site Seq est Kd [nM] # exact matches Hamming Dist 1 Hamming Dist 2 Hamming Dist 3
BBa_J05100 ggaggggac 4 5 267 6196 65733
BBa_J05102 ggaggcggg 30 17 799 12589 95996
BBa_J05101 gggggcgag 3 15 634 10173 82956
BBa_J05103 ggggccgga 45 44 848 11317 91422
BBa_J05108 gtcccctccggaggggac N/A 0 0 0 1
BBa_J05109 ctcgcccccgggggcgag N/A 0 0 0 2
BBa_J05110 cccgcctccggaggcggg N/A 0 0 0 4
BBa_J05111 tccggccccggggccgga N/A 0 0 0 6
BBa_J05115 gggggcgaggggggcgag N/A 0 0 0 3


Degradation rates????

Tests: design all tests. To test whether our assumptions about using multiple zinc finger proteins (ZFP) as repressors (i.e. roadblocks) will actually work, we will build a tester/debugging device in parallel with the counter.


BBa_J05000 Zinc Finger Protein Tester
Repressor Regulator Comments
BBa_J05112 R3-e2+ERD Regulator for R3-ATF6
BBa_J05113 R3-ATF6+ NTD BBa_J05221 Triple Binding Site for R3-ATF6
BBa_J05114 R3-ATF6+NTD Regulator for R3-ATF6
BBa_J05101 R3-e2 Regulator for R3-ATF6
BBa_J05115 ZF-2*e2 ZF-2*e2 Binding Site


assembly diagram of test. TesterParts.gif


4SD system The 4SD system consists of 4 proteins with 4 operator regions. It has an interface boundary with input module (Pr and Prm). Repressor R3 is connected to a reporter to be able to count to modulo two. assembly diagram of device. CounterParts.gif

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