4-State Device
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Contents |
Purpose
The 4-State Device (4-SD) uses two inputs to sequentially switch through its four states. The four states are used to keep of changes of the inputs. One state is connected to the output.
Design
Suitable Strategies
we need repressors...
we have considered: existing promotors, RNA interference, ZFP
Selected System
ZFP A Zinc Finger (ZF) [1] is a protein domain [2] 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. why. for each state we need one repressor protein. modular design. won't run out of Repressors. 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). Another important aspect is the overall goal of keeping the design modular - one of the most important aspects of the this contest.
how.
The central part of the counter 4SD. When input changes the next state is reached and the repressor is expressed. Every Repressor represses all other repressors than the next. For example R1 represses R3 and R4 and in the next cycle R2 forced.
Actual Implementation
In the figure below is a parts-view of the device with PoPS interfaces (i/o).
domains: ZF NTP ERD, etc. 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 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. An alternative could be a negative transcription domain fused to the ZFP.
table of parts.
We use several different designs of ZFPs.
- Three ZF domains to recognize nine base pairs.
- 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.
- Six ZF domains to recognize twelve base pairs.
- Three ZF domains fused to negative transcription factors. (e.g. NTD and ERD)
| Nickname | Length | SacI | KpnI | NheI | EcoRI | XbaI | SpeI | PstI |
|---|---|---|---|---|---|---|---|---|
| BBa_J05215+BBa_J05108 | $874.35 | 1 | 91 | 598 | 7, 97 | 22, 112 | 71, 578 | 85, 592 |
| BBa_J05216+BBa_J05109 | $843.9 | 1 | 91 | 577 | 7, 97 | 22, 112 | 71, 557 | 85, 571 |
| BBa_J05217+BBa_J05110 | $883.05 | 1 | 91 | 604 | 7, 97 | 22, 112 | 71, 584 | 85, 598 |
| BBa_J05218+BBa_J05111 | $870 | 1 | 91 | 595 | 7, 97 | 22, 112 | 71, 575 | 85, 589 |
| BBa_J05221+BBa_J05101 | $722.1 | 1 | 112 | 493 | 7, 118 | 22, 133 | 92, 473 | 106, 487 |
| BBa_J05114 | $1238.3 | 1 | 849 | - | 7 | 22 | 829 | 843 |
| BBa_J05112 | $759.8 | 1 | 519 | - | 7 | 22 | 499 | 513 |
| BBa_J05222+BBa_J05115 | $980.2 | 1 | 87 | 671 | 7, 93 | 22, 108 | 67, 651 | 81, 665 |
describe the trick we use to reduce cost, that is, putting small parts together with big parts.
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 the this contest.
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.
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 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.
| Part | Binding Site Seq | # exact matches | Hamming Dist 1 | Hamming Dist 2 | Hamming Dist 3 |
|---|---|---|---|---|---|
| BBa_J05100 | ggaggggac | 1 | 132 | 3125 | 32788 |
| BBa_J05102 | ggaggcggg | 5 | 396 | 6293 | 47897 |
| BBa_J05101 | gggggcgag | 8 | 303 | 4955 | 41302 |
| BBa_J05103 | ggggccgga | 23 | 433 | 5606 | 45492 |
| BBa_J05108 | gtcccctccggaggggac | 0 | 0 | 0 | 0 |
| BBa_J05109 | ctcgcccccgggggcgag | 0 | 0 | 0 | 2 |
| BBa_J05110 | cccgcctccggaggcggg | 0 | 0 | 0 | 3 |
| BBa_J05111 | tccggccccggggccgga | 0 | 0 | 0 | 3 |
| BBa_J05115 | gggggcgaggggggcgag | 0 | 0 | 0 | 1 |
Kd values of interactions. put in table above.
Degradation...?
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.
| Repressor | Operator | Comments |
|---|---|---|
| J05100 | J05212 | ... |
| J05100 | J05212 | ... |
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.
