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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 ...	+
-	===Meetings===	+	== Suitable Strategies ==
-	'''2005.08.17, Wednesday, 15:30 @ Alex' bureau (CAB F62)''' [[Summary]]	+	For realizing the design we need one repressor for each state. For this purpose there are several possibilities that can be considered:
-	'''2005.08.18, Thursday, 13:15 @ Alex' bureau (CAB F62)'''	+
-	'''2005.08.22, Monday, 13:45 @ Alex' bureau (CAB F62)''' [[Summary 050822]]	+
-	'''2005.08.25, Thursday 15:30 @ Alex' bureau (CAB F62)'''	+
-	'''2005.08.29, Monday 14:00 @ Alex' bureau (CAB F62)'''	+
-	'''2005.08.30, Tuesday 16:00 @ Alex' bureau (CAB F62)'''	+
-	'''2005.09.06, Tuesday 08:00 @ Alex' bureau (CAB F62)'''	+
-	=Description of 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-module Schematic==	+	* Existing Repressor-Operator (for example TetR-TetO)
-	Below a preliminary parts-view of the module with PoPS interfaces (i/o).	+
-	---------------------------------	+	* Designing new repressors (for example Zinc Finger Proteins)
-	|  ------------\                |	+
-	PoPS_in1 ---->|  | Repressor1 | --------      |	+
-	|  ------------/          |    |	+
-	|                        =    |	+
-	|                      --------|------> PoPS_out	+
-	|                        =    |	+
-	|  ------------\          |    |	+
-	PoPS_in2 ---->|  | Repressor2 | --------      |	+
-	|  ------------/                |	+
-	---------------------------------	+
-	=Design=	+	== 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.
-	=== Zinc Fingers ===	+	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).
-	A zinc finger [http://en.wikipedia.org/wiki/Zinc_finger]  is a protein domain that binds to double stranded DNA. The idea is to use a zinc finger protein (ZFP) as a repressor by putting a ZFP binding site upstream of the coding region and thereby preventing RNA polymerase to transcribe the gene. A ZFP consistists of one or several Zinc Finger Domains that binds to three base pairs each. Many domain-DNA interaction for a triplet of base pairs have been described, therefore making it possible to to construct artificial transcription factors by combining domains.	+
-	===== Zinc Finger Proteins =====	+	The silencing approach has been dropped because of the lack of modularity and the difficulty to achieve full repression via silencing approaches in bacteria.
-	In total for the counter we will need four ZFPs. We have two different designs of the ZFPs.	+
-	1. Three domains to recognize nine base pairs.	+	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.
-	2. two zinc finger 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.	+	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.
-	===== Zinc Finger Protein Binding Sites =====	+	= Actual Implementation =
-	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.	+	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.
-	We have to different candidates for placing the binding sites.	+
-	1. Binding sites in the promoter. This would prevent the polymerase from binding to the promotor.	+	In the figure below is a parts-view of the device with PoPS interfaces (i/o).
-	2. Binding sites directly after the start of transcription and before the ribosome binding site. This alternative is quite 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.	+	==== 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.
-	===Tester for Zinc Finger===	+	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>
-	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 .	+
-	===System Variants===	+	===== Electrical NOR gate =====
		+	A NOR gate is a very common part in electrical engineering.
-	Currently, we have two systems, and we hope that at least one of them is going to work.	+	[[Image:NORcircuit.gif]]
-	The first system, cryptically labelled "3dZFp-R" below, consists of 4 proteins with 4 operator regions. Each protein has four domains: a) 3 zink fingers that bind to specific sites on the helical DNA, and b) one repression (R) domain. We call the whole system (i.e. a+b) "Zinc Hand" (ZH) to distinguish it clearly from the individual zinc finger domains.	+	===== 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)
-	In the second system, labelled "2dZFp-LZ-R" (LZ = leucine zipper), each protein has also 4 domains: 2 zink fingers, 1 leucine zipper (LZ) and 1 repression (R) domain.	+	[[Image:NOR.gif]]
-	The operator regions in both systems are made of 2 binding regions, each of which lets a specific Zinc Hand bind to it and thus each consisting of the corresponding 3 codons that match the 3 zinc fingers in that Zinc Hand. The operator regions (i.e. the "roadblocks" that will prevent the binding of the RNApolymerase) form a BioBrick that should be inserted between the promoter region and the ribosome binding site in order to keep the design modular. Since we always use 2 Zinc Hands to block a promoter site (i.e. the promoters Pr and Prm of the lambda-system) and since we have 4 such promoter sites (i.e. 2xPr and 2xPrm), we need to have 4 operators = 4 permutations of Zinc Hand pairs.	+	===== 4-State Device circuit diagram =====
		+	[[Image:4SDcircuit.gif]]
-	ZHR stands for '''Z'''ink '''H'''ands '''R'''egulatory proteins, and ZHO for '''Z'''ink '''H'''ands '''O'''perators. The +1/+10 indication reflects the starting position of the 2 binding regions on the DNA. In both case, it between the promoter region (<0) and the ribosome binding site (around +20). There has been some discussion about having the operator around 0 (-9/+1)	+	===== 4-State Device parts diagram =====
-	{| border="1"	+	[[Image:4StateDevice.png]]
-	! || Type || Size (bp) per Unit || . || . || . || . || TOTAL SYSTEM SIZE (bp)	+
-	|-	+	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.
-	!3dZFp-R	+
-	| Repressor (protein) || 400 || ZHR1 || ZHR2 || ZHR3 || ZHR4 || 1600	+	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
		+	{| border="1" cellpadding="2"
		+	|+ Synthesized Sequences
		+	! BioBrick Part !! Number of ZF domains !! LZ domain !! TF domain
	|-		|-
-	| . || Operator +1/+10 (target sequence) || 30 || ZHO1.1 || ZHO2.1 || ZHO3.1 || ZHO4.1 || 120	+	| BBa_J05101 || 3  || -    ||   -
-	|-	+
-	!2dZFp-LZ-R	+
-	| Repressor (protein) || 400 || ZHR5 || ZHR6 || ZHR7 || ZHR8 || 1600	+
	|-		|-
-	| . || Operator +1/+10 (target sequence) || 30 || ZHO5 || ZHO6 || ZHO7 || ZHO8 || 120	+	| BBa_J05108 || 3* || CREBH || -
	|-		|-
-	! TOTAL || . || . || . || . || . || . || 3440	+	| 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  || -    || -
	|}		|}
-	====Repression====
-	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:
-	* 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====	+	==== BioBrick parts ====
-	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.	+
-	====Additional Comments====	+	{| border="1" cellpadding="2"
-	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 simple won't have the time for it.	+	|+ BBa_J05040 4-State Device
-	Another important aspect is the overall goal of keeping the design modular - one of the most important aspects of the this contest.	+	! State !! Repressor  !! Regulator !! Comments
-	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).	+	|-
		+	| R1 || BBa_J05311 || BBa_J05215 ||
		+	|-
		+	| R3 || BBa_J05312 || BBa_J05216 ||
		+	|-
		+	| R2 || BBa_J05313 || BBa_J05217 ||
		+	|-
		+	| R4 || BBa_J05314 || BBa_J05218 ||
		+	|}
-	===Assembly===
-	* 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
-	* ...
-	(please complete the above and add the test/debugging steps)
-	=References=	+	{| border="1" cellpadding="2"
-	[[Kim & Wang]] (??)	+	|+ 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
		+	|}
-	[[Park et al.]] 2005 (Activation of transcription)
-	[[Chou et al.]] 1998	+	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.
-	[[Mani05]]	+	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.
-	[[Beerli98]]
-	[[Beerli00]] (Linker)
-	[[Beerli02]]	+	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.
-	[[Isalan01]]	+	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.
-	[[Greisman97]]	+	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.
-	[[Segal03]]	+	{| 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
		+	|}
-	[[Segal99]] ("GNN-paper")
-	[[Dreier01]] ("ANN-paper")
-	[[Dreier05]] ("CNN-paper")	+	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.
		+
		+
		+	{| 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")
-	=Design=	+	4SD system
-	==Overall Design==	+	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.
-	[[Image:OverallDesign.png.png]]	+	assembly diagram of device.
-	==What to order==	+	[[Image:CounterParts.gif]]
-	[[Image:WhatToOrder.png.png]]	+

---

Latest revision as of 19:58, 5 November 2005

Back to the ETH Zurich main page.

Contents 1 Purpose 2 Design 2.1 Suitable Strategies 2.2 Selected System 3 Actual Implementation 3.1 Basic Functionality 3.1.1 Electrical NOR gate 3.1.2 Biological NOR gate 3.1.3 4-State Device circuit diagram 3.1.4 4-State Device parts diagram 3.2 Biological Details 3.2.1 BioBrick parts

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.

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)

4-State Device circuit diagram

4-State Device parts diagram

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.

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.