Background

Cells are responsive to a myriad signals under most conditions[1]. Upon receiving this cornucopia of signals, i.e. inputs from the external and internal environment, cells must adjust their own internal mechanisms in response in order to survive. This adjustment depends not only on processing a combination of current environmental signal inputs[1], but also on determining the cell’s current state, which is a result of a series of past inputs. In dig-ital circuit theory, this operating mode is known as sequential logic[3]. Sequential logic is a type of logic in which output is a function of the present value of inputs and the system’s internal state.

The field of synthetic biology aims to design biological systems to perform tasks to better understand analogous natural systems and to develop direct applications for research and medicine. Nowadays, a wide va-riety of tasks can be performed by synthetically engineered genetic circuits, mostly constructed using combinational logic. The output of combinational logic (also referred to as time-independent logic) is a function of the present input only[4]. In contrast to sequential logic, it has no internal state as an influence of the history of inputs. Without a memory of state, it is difficult to perform functions in a specific order, which has limited the widespread implementation of such systems[5].

The ability of sequential logic circuits to store modest amounts of information within living systems and to act upon them would enable new ap-proaches to the study and control of biological processes[6]. Indeed, the attention paid to this field has progressed far beyond scarce. For example, genetic toggle switches have proved to be effective in building synthetic memory modules[7]. In 2010, a bistable push-on push-off switch was constructed as a memory module by Lou et al[8][9]. Five years later, Pakpoom Subsoontorn et al. designed a toggle flip-flop switch whose state can be changed by the same signal[10]. Moreover, in 2012, Jerome Bonnet demon-strated a rewriteable recombinase-addressable data (RAD) module[11]. Re-cently, a state machine with two inputs and five states depending on the order of the input was realized in E. coli by Roquet, N. et al[12].

These studies indicate that more functions could be accomplished with a memory module embedded within the circuit[13]. Once the memory system is able to scale up to more states, a cell can be designed to do work that is more complex. In other words, we can reach a new dimensionality in designing synthetic life–time.

Introduction

This year, the Peking iGEM team is attempting to develop a frame of biological sequential circuits that are programmable. The envisioned circuit is capable of both storing states in DNA and automatically running a series of instructions in a specific order. More specifically, the sequential logic (or a real-state machine) that consists of a clock (trigger signal), flip flop (remembering device) and control unit (functional part) in bacteria. The clock is an oscillator with a repeated signal cycle that is utilized like a metronome to trigger actions of sequential logic circuits. Flip-flop is a memory device that can remember a state. Paired with a clock signal, it can realize state transition. The control unit is a functional part which can convert a signal from flip-flop into complex functions. With such a design, historical events are recorded and influence current cell behavior.

This work tries to point the way toward building large computational systems from modular biological parts—basic sequential computing devices in living cells, and ultimately, programming complex biological functions. Computers have thus become "alive". A unicellular organism itself cannot pack much computational power, but considered as a modular building block, its potential is impressive.

References

Hansen, J., & Benenson, Y. (2016). Synthetic biology of cell signaling. Natural Computing, 15(1), 5-13.
Densmore, D., & Anderson, J. C. (2009, May). Combinational logic design in synthetic biology. In Circuits and Systems, 2009. ISCAS 2009. IEEE International Symposium on (pp. 301-304). IEEE.
Cavanagh, Joseph (2006). Sequential Logic: Analysis and Synthesis. CRC Press. pp. ix. ISBN 0849375649.
C.J. Savant, Jr.; Martin Roden; Gordon Carpenter. "Electronic Design: Circuits and Systems". 1991. ISBN 0-8053-0285-9 p. 682
Lou, C. et al. Synthesizing a novel genetic sequential logic circuit: a push-on push-off switch. Mol. Syst. Biol. 6, 350 (2010).
Zhang, H., Lin, M., Shi, H., Ji, W., Huang, L., Zhang, X., ... & Yang, Z. (2014). Programming a Pavlovian-like conditioning circuit in Escherichia coli. Nature communications, 5, 3102.
Subsoontorn P, Endy D (2012) Design and analysis of genetically encoded counters. Procedia Comput Sci 11:43–54. doi:10.1016/j.procs.2012.09.006CrossRefGoogle Scholar
J. Bonnet, P. Subsoontorn, D. Endy, Rewritable digital data storage in live cells via engineered control of recombination directionality. Proc. Natl. Acad. Sci. U.S.A. 109, 8884–8889 (2012). 10.1073/pnas.1202344109pmid:22615351 doi:10.1073/pnas.1202344109
Roquet, N., Soleimany, A.P., Ferris, A.C., Aaronson, S. & Lu, T.K. Synthetic recombinase-based state machines in living cells. Science 353, aad8559 (2016).
Burrill, D. R., & Silver, P. A. (2010). Making cellular memories. Cell, 140(1), 13-18.

Back to top

Flip-flop

Background

The basic building blocks of sequential logic circuits are flip-flops, which are devices that can store information. A flip-flop has two stable states (see more on "state") and its state can be changed by signals of arriving pulses. Thus, it and can store one bit of information and alternate its state following a trigger. It is the basic storage element in sequential logic. Click here to get a more specific description

When designing genetic-circuit counterparts of flip-flops, we should consider the following three criteria:

The device should have at least two stable states;
State changes are triggered by an identical signal;
The flip-flop should hold its state and remain unaltered until the next signal arrives.

In a living cell, DNA is the natural medium for storing cell-state information and encoding functions. Recombinases, especially a subset called serine integrases and excisionases, are enzymes that can flip or excise specific fragments of DNA. They have been proved to be able to stably modify DNA sequences , which is the biological basis of our genetic flip-flop construct.

Large serine integrases reliably and irreversibly flip or excise unique fragments of DNA . DNA cleavage and re-ligation occur at the central crossover region at a pair of recombinase recognition sites (attB and attP), which allows the sequence to be flipped, excised, or inserted between recognition sites . After recombination, the original attB and attP sequences become reconstructed sequences - attL and attR. The resulting attL and attR sequences cannot be recognized and bound by integrases alone, so the state after integration is stable.

Another kind of recombinases, excisionases, are able to recognize and bind attL and attR sequences. With the help of excisionases, the state transitions become reversible.

Recombination Directionality Factors (RDF) are proteins which mediate reverse flipping to recover the sequence flipped by recombinases. By co-expressing the integrase with the corresponding RDF, or expressing the integrase-RDF fusion protein, recombination between the attL and attR sites can be induced. Thus, the flip-flop can be restored to the previous state.

Figure 1. Site-specific recombination either integrates, deletes or reverses a DNA sequence.

Aims

We would like to prove the feasibility of bio-flip-flops and optimize their function as parts. In order to do this, we have to:

Select effective recombinases and their corresponding RDFs; characterize and optimize their function.
Construct half of the flip-flop structure and express it in E. coli.

We would first search for known recombinase sequences in the literature, construct the corresponding vectors, and select the best ones to make standardized parts. After confirming their expression and function, we would optimize these devices through the standard workflow of vector and RBS substitution. We would also prove integrase-RDF fusion protein function. Furthermore, we would con-struct half of the bio-flip-flop structure and experimentally show that it can function.

This would serve as a proof of concept for a novel design strategy for bio-flip-flops.

Key Achievements

Test and optimize recombinase and recombinase-RDF fusion protein function.
Standardization into BioBrick devices.
Introduction of the forward latch of the bio-flip-flop into cells and proof of function in vivo.

Our Approach and Designs

Design of the bio-flip-flop

Using a pair of integrases and their corresponding excisionases, we designed the bio-flip-flop device to store state information as follows:

The device has two promoters, controlled by input inducers X and Y, respectively. The input can either promote or inhibit transcription.
In the schematic mechanism figure below, recombination sites with colors corresponding to their binding integrases are placed on both sides of the promoter in inverse directions. Thus, integrases can flip the promoters, changing the direction of transcription.
We define the combination of the input series X, and then Y as a single signal. Thus, the bio-flip-flop can change the circuit state each time a signal arrives, and keep this state until the next signal pulse.

Figure 2. Schematic diagram of the bio-flip-flop mechanism.

Selection of Available Integrases

We first did a literature search and sequence alignment to mine the databases for available integrases for device development. Here we list the names and sources of the collected recombinases.

We finally chose to use TP901-1, Bxb1 and phiC31. These are well-known serine integrases, and all of them have been used to demonstrate static-input logic gates. Some have cofactors (equivalent to RDFs) that can reverse directionality. Previous work has shown that there exists a new set of 11 orthogonal integrases, greatly expanding the availability of integrases that can be formed into standardized parts. We hope that given enough time and resources, it is possible to construct more parts using our standard workflow described below.

Standard Characterization & Optimization Workflow

The viability of a bio-flip-flop relies on the performance of two integrases and their corresponding excisionases. To select integrases for the bio-flip-flop, we constructed expression vectors for different recombinases and tested their performance individually. Here we introduce a standard workflow to accomplish this task.

Figure 3. The workflow of bio-flip-flop optimization.

Different replication origins affect the copy number of plasmids, and it is highly probable that they also influence the expression levels of their encoded recombinases. To select integrases for the bio-flip-flop, we constructed expression vectors for different recombinases and tested their performance individually. This was carried out by co-transformation of E. coli Top10 with the expression vector and a reporter plasmid. The reporter plasmid expresses differen t fluorescent proteins before and after flipping of a constitutive promoter.

Figure 4. The standard genetic structure used to characterize the recombinases.

According to our design of the bio-flip-flop, the two inputs X and Y comprise a signal. Consequently, two distinct induction systems need to be constructed. For this reason, recombinases were cloned onto two different backbones, under regulation of differently induced promoters. The expression vectors were constructed by Gibson assembly. Plasmid type 1 is cloned from the repressor generator (RPG), which has a p15A replication origin, ampicillin resistance gene, lacI, an IPTG-induced pTac promoter, and a RiboJ insulator after pTac. The backbone of plasmid type 2 was cloned from pIntegrase_13, which was a gift from Christopher Voigt (Addgene plasmid # 60584). It has a ColE1 replication origin, kanamycin resistance gene, araC, and an arabinose-induced pBad promoter. RiboJ was added to Plasmid2 by cloning RiboJ and the recombinase from Plasmid1, and assembled after pBad.

Figure 5. Integrase expression vectors with different replication origins. The one shown on top has a relaxed ColE1 replication origin and recombinase expression is induced by arabinose via a pBad induction system. The one shown in the bottom picture has a relaxed p15A replication origin and recombinase expression is induced by IPTG.

For a given backbone and inducible promoter, RBS strength affects the expression level post-transcription. For this reason, we tested a series of RBS for different recombinases in different backbones.

In order to tune the RBS, an intermediate vector with a 20bp random sequence (45% GC), flanked by a pair of BsmbI recognition sites (introduced by PCR) was prepared for each recombinase. Vector construction was completed by adding RBS via Golden Gate assembly. The RBS calculator was used to generate RBS sequences with a wide range of translation initiation rates. Other sequences (B0029, B0030, B0032, B0034, B0035) were obtained from the iGEM website. The RBS oligos were obtained from the annealing of two complementary primers. Expression vectors for each recom-binase with different RBS were tested by flow cytometry after co-transformation with the reporter vec-tor. For each recombinase, an RBS correlating with low leakage (small RFP subset in the cultures with no inducer) and high recombination efficiency after induction was selected.

Measurement of Recombination Efficiency

We used a microplate reader to roughly measure the efficiency of the selected integrases. We used flow cytometry to conduct a more accurate characterization.

Microplate Readers

Microplate readers are instruments used to detect biological, chemical or physical events in samples in microtiter plates. We used a microplate reader to detect optical density and fluorescence intensity follwing the procedure as below:

Pick and inoculate single colony into 1ml of LB media with antibiotics in a V-bottom 96-well plate. Grow the culture overnight (about 12 hours) at 37°C, 220 rpm in a Thermo VARIOSKAN FLASH shaker.
Then, an aliquot comprising 2 μL of the culture was transferred into 1ml LB media with antibiotics and inducer (1mM IPTG or 10mM arabinose). Culture overnight (about 12 hours) at 37°C and 220 rpm.
Aliquot 1 μl of the cultures in the deep-well plates, then copy them to the 96-well plate filled with 200μl PBS. Don’t forget to leave at least 12 wells with only PBS in it as your negative conctrol.
Measure the samples (OD and Fluorescence). The former represents the density of bacteria, and the latter implies the efficiency of recombination. GFP/OD is calculated to measure recombination efficiency.

Flow Cytometry

For more detailed measurements,flow cytometry were used to evaluate the recombination efficiency. Our procedures are as follows:

Single colonies were picked and used to inoculate into 1ml of LB media with antibiotics in a V-bottom 96-well plate.
The cultures were grown at 37°C and 1000 RPM for 12h. Subsequently, an aliquot comprising 2 μL of the culture was transferred into 1ml of M9 glucose media with antibiotics and inducer (1mM IPTG or 10mM arabinose for RBS tuning, gradient concentration for transfer curve) in a V-bottom 96-well plate.
The cultures were grown at 37°C and 1000 RPM for 15h. An aliquot comprising 2μL of the culture was transferred into 198 μL of phosphate buffered saline (PBS) containing 2 mg/mL kanamycin in a 96-well plate. This mixture was incubated for one hour at room temperature before testing. Two lasers were used to excite GFP and RFP simultaneously. Single-cell fluorescence distribution at both emission wavelengths was recorded.

The counted cells were gated to eliminate the population which showed no fluorescence. The remaining cells were divided into two subsets by a diagonal: RFP sub-set and GFP subset. The recombination efficiency was estimated from the proportion of the RFP subset in the total fluorescent population.

Figure 6. A example of Gating the RFP and GFP subsets. Change of fluorescence after induction was seen. Left: no inducer. Right: 10 mM Ara for 15h. Change of fluorescence after induction was seen. Left: no inducer. Right: 10 mM Ara for 15h.

For the vector with ColE1 replication origin, we found proper RBS in a list of calculated RBSs for TP901-1.

We observed a shift in fluorescence level and wavelength in several trails, which indicated that recombinase can flip the promoter region and alter gene expression in our design. However, problems such as leakage and insufficient recombination efficiency were obvious. Tuning of our expression system seemed necessary before assembly into the bio-flip-flop device.

Characterization of Excisionases

To make sure that the sequence-reversing process is efficient and complete, we used two strategies: one encompasses combining the integrase and its corresponding RDF into a polycistronic structure, and the other is to construct fusion proteins comprising the integrases and RDFs in one polypeptide chain. We have been trying to improve the recombination efficiency by replacing the expression vec-tor with different replication origins or inducible promoters and changing the RBS sequence before the coding sequence.

Figure 7. The two strategies used to characterize integrases and their RDFs. UP. The polycistronic structure containing the integrase and its corresponding RDF. Bottom. The fusion protein structure of integrase with its corresponding RDF.

The method is similar to the integrase test method. We constructed an input plasmid to express integrases and RDFs, and an output plasmid on which the promoter J23119 is located between the attL and attR sequences. The input plasmid expresses GFP before induction of integrase and RDF, and if an inversion occurs after induction, the output plasmid can express RFP. By comparing the changes in fluorescence types and intensities before and after induction, the efficiency of inversion can be estimated.

Figure 8.The standard genetic structure used to characterize the recombinases.

Construction of Bio-flip-flops

After having already optimized the expression systems of integrases and excisionases, we set out to construct a Bio-Flip-Flop. Since it is unknown whether the Bio-Flip-Flop is practical, we considered developing two hierarchical execution units supporting our Bio-Flip-Flop. We called these two units the forward latch and the backward latch. The state transition process of each unit consists of two stable and irreversible states. Nevertheless, the state transition proces s of both units consists of four stable and cyclic states. Such units support the verification of the feasibility of our Bio-Flip-Flop and further allow us to construct a complete and functional Bio-Flip-Flop.

Forward Latch

The structure of the forward latch consists of two plasmids with different induction systems of integrases (Figure 9). The state transition process of the forward latch consists of two stable and irreversible states (Figure 10).

Figure 9. The structure of the forward latch. Two plasmids constitute the forward latch. Each plasmid contains an integrase gene (Bxb1-gp35 or TP901) and a reporter gene (GFP or mRFP). To standardize the input, we used the pBAD promoter to regulate the transcription of Bxb1 and GFP, and the pTAC promoter to regulate the transcription of TP901 and mRFP. Moreover, each integrase recognizes and converts the attB and attP sites on the other plasmid.

Figure 10. The state transition process of the Forward latch. After induction with arabinose, the expression of integrase Bxb1 and GFP is initiated. The integrase Bxb1 recognizes and converts the attB and attP sites flanking the pTAC promoter, which leads to a change of orientation of the pTAC promoter. If we input IPTG next, the expression of integrase TP901 and mRFP will begin. The orientation of the pBAD promoter will change. As the GFP is degraded and mRFP is produced, the ratio of mRFP/GFP fluorescence rises.

Backward Latch

The structure of the Backward latch consists of two plasmids with different induction systems driving excisionases (Figure 11). The state transition process of the Backward latch consists of two stable and irreversible states (Figure 12). Because excisionases recognize attL and attR sites and convert them into attB and attP sites, the backward latch is capable of resetting the state of the Forward latch.

Figure 11. The structure of the Backward latch. Two plasmids constitute the Backward latch. Each plasmid contains an excisionase gene (Bxb1-gp47 or TP901) and a reporter gene (GFP or mRFP). To standardize the input, we used the pBAD promoter to regulate the transcription of Bxb1 and GFP, and the pTAC promoter to control the transcription of TP901 and mRFP. Moreover, each excisionase recognizes and converts the attL and attR sites on the other plasmid.

Figure 12. The state transition process of the Backward latch. After induction with arabinose, the expression of excisionase Bxb1 and GFP is initiated. The excisionase Bxb1 recognizes and converts the attL and attR sites flanking the pTAC promoter, which leads to a change of the orientation of the pTAC promoter. If we input IPTG next, the expression of excisionase TP901 and mRFP will begin. The ori-entation of the pBAD promoter will change. As GFP is degraded and mRFP is produced, the ratio of mRFP/GFP fluorescence rises.

Experiment

Because our final goal is to realize the state transition of our Bio-Flip-Flop, it is significant to characterize the efficiency of the primary state transition of the forward latch. We quantified the amounts of GFP and mRFP and considered the ratio of mRFP/GFP fluorescence as a direct measure of the efficiency of the state transition.

For all characterization experiments, co-transformed cells were first grown and subcultured overnight, after which we induced the cells with arabinose and IPTG in that order. Finally, we quantified the fluorescence strength using a flow cytometer.

Results

Primary Proof of Recombination

To prove the function of our integrase-RDF fusion protein in an illustrative way, IPTG was applied to the petri dish where E. coli Top10 co-transferred with expression and reporter plasmid were cultured. Compared to the RFP control (transferred with RFP expressing reporter plasmid only), green fluorescence was obvious when excited by visible light. Half red, half green colonies were seen, indicate incomplete recombination.

Figure 13. (left) RFP control. (middle) expression vector of TP901-RDF fusion protein co-transferred with reporter. White arrows: colonies with green fluorescence indicates recombination. (right) A example of half red, half green colonies, indicating incomplete recombination.

In a similar trail, Arabinose was applied to the petri dish where E. coli Top10 co-transferred with TP901-1 integrase expression and reporter plasmid were cultured. Compared to the GFP control (transferred with GFP expressing reporter plasmid and expression vector, but no inducer was added), We can see a transition of colony color from green (GFP expressin) to red (RFP expression). When excited by visible light, it’s distinguishable that the colonies nearly completely cease to express GFP. The expression of RFP and shut down of GFP expression shows that recombination has happened.

Figure 14. (Up, left) GFP control, colonies in green. (Up, right) GFP control, green fluorescence can be seen when excited. (Bottom, left) Expression vector of TP901-1 integrase co-transferred with reporter, red color instead of green indicates recombination. (Bottom, right) Expression vector of TP901-1 integrase co-transferred with reporter, green fluorescence is nearly completely shut down, indicating desirable recombination efficiency.

Fine-tuning of Recombination Efficiency

Selection of RBS for different replication origin

For the vector with ColE1 replication origin, we found proper RBS in a list of calculated RBSs for TP901-1 and Bxb1 gp35.

Figure 15. Bxb1 gp35 recombination efficiency with a variety of calculated RBS and RBS sequences from iGEM (B0030∼B0035). 
T.I.R = Translation Initiation Rate

Figure 16. TP901-1 recombination efficiency with a variety of calculated RBS. A. At the vector with p15A. 
B. At the vector with colE1. 
T.I.R = Translation Initiation Rate 

For the expression vector with p15A replication origin, a proper RBS for TP901-1 was selected.

Figure 16_b. TP901-1 recombination efficiency with a variety of RBS from iGEM (B0030∼B0035). 
T.I.R = Translation Initiation Rate

Transfer curves

We evaluated the performance of phiC31 and Bxb1 gp35 on ColE1 expression vectors under a series of inducer concentration gradients. The results enabled us to determine the appropriate inducer concentration.

A

B

Figure 17. The transfer curves of the recombinases we utilized. A. The transfer curve of integrase phiC31. B. The transfer curve of integrase Bxb1.

Measurement of the Flipping Efficiency of Excisionases

We have tested the flipping efficiency of our Integrase-RDF fusion proteins with a group pf RBS. Flipping performance varies with different RBS. For TP901-RDF fusion proteins, recombination seems not obvious in some cases (RBS B0032, B0034, B0035, phiC 10000, Bxb 1000, int2 3000), while in others both flipping efficiency and leakage and significant.

Figure 18. pTac TP901-RDF fusion protein flipping efficiency with ten RBS. Proportion of GFP subset indicates flipping efficiency.

A second test was supplemented. Similar results support that our Integrase-RDF fusion proteins are able to flip the sequence between attL and attR site of the reporter, but much improvement are needed to reduce the leakage as well as increase the efficiency.

Figure 19. pTac TP901-RDF fusion protein flipping efficiency with two RBS. Proportion of GFP subset indicates flipping efficiency.

Integrase-RDF for Bxb1 and phiC31 were also constructed, tested. High flipping efficiency and leakage were seen.

Figure 20. Flipping efficiency for pTac integrase-RDF fusion protein of phiC31 and Bxb1. Proportion of GFP subset indicates flipping efficiency.

To conclude, we found that the Integrase-RDF fusion proteins are able to flip the sequence between attL and attR site (indicated by observed GFP subset), although tuning work remains to be done to construct a controllable, functional bio-flip-flop device.

Reference

Yang, L., Nielsen, A. A., Fernandez-Rodriguez, J., McClune, C. J., Laub, M. T., Lu, T. K., & Voigt, C. A. (2014). Permanent genetic memory with> 1-byte capacity. Nature methods, 11(12), 1261-1266.
Bonnet, J., Yin, P., Ortiz, M. E., Subsoontorn, P., & Endy, D. (2013). Amplifying genetic logic gates. Science, 340(6132), 599-603.
Baker, T. A., Bell, S. P., Gann, A., Levine, M., & Losick, R. (1970). Molecular biology of the gene.
Olorunniji, F. J., McPherson, A. L., Rosser, S. J., Smith, M. C., Colloms, S. D., & Stark, W. M. (2017). Control of serine integrase recombination directionality by fusion with the directionality factor. Nucleic acids research, 45(14), 8635-8645.
Siuti, P., Yazbek, J., & Lu, T. K. (2013). Synthetic circuits integrating logic and memory in living cells. Nature biotechnology, 31(5), 448-452.
Khaleel, T., Younger, E., McEwan, A. R., Varghese, A. S., & Smith, M. (2011). A phage protein that binds φC31 integrase to switch its directionality. Molecular microbiology, 80(6), 1450-1463.
Salis, H. M., Mirsky, E. A., & Voigt, C. A. (2009). Automated design of synthetic ribosome binding sites to control protein expression. Nature biotechnology, 27(10), 946-950.
Espah Borujeni, A., Channarasappa, A. S., & Salis, H. M. (2013). Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic acids research, 42(4), 2646-2659.

T Flip-Flop

Basically, a real flip flop has two inputs. One input is a control input. For a T flip flop, the control input is labelled T. The other input is the clock. Positive edge-triggered flip flops can only change output values when the clock is at a positive edge. While negative edge triggered flip flops change on a negative edge, and level-triggered flip flops, that change only when the value is 1. When the clock is not at a positive edge, then the output value is held. That is, it does not change. A flip flop’s output is the bit it stored. Thus, the flip flop is always outputting the one bit of information.
Here's the characteristic table for a T flip flop.

T	Q	Q+	Operation
0	0	0	Hold
0	1	1	Hold
1	0	1	Toggle
1	1	0	Tohgle

The T flip flop characteristic table has 3 columns. The first column is the value of T, a control input. The second column is the current state, that is the current value being output by Q. The third column is the next state, that is, the value of Q at the next positive edge. It's labelled with Q and the superscript, + (the plus sign).

The T flip flop has two possible values. When T = 0, the flip flop does a hold. A hold means that the output, Q is kept the same. When T = 1, the flip flop does a toggle, which means the output Q is negated. Thus, in a T flip flop, you can either maintain the current state's value for another cycle, or you can toggle the value (negate it).

Why isn't it XOR?
First, XOR has two inputs, and one output. A T flip flop essentially has a control input and a control output. Second, the second column and the third column are really the same output, but at different points in time.

Difference between Flip flop and gate
The biggest difference between a flip flop and a gate is that a flip flop can hold its value. Even though holding a value is something very simple, it makes it different from logic gates, and allows us to design circuits that have cycles in them (i.e., feedback).

State

There is a generic definition of state:
State is a quantity that stores the previous history of what's happened in such a way that you can predict the output, given any future input.

From the definition, we can get two things. First, state refers to some recorded information. We can consider state as a kind of memory. For sequential logic circuits, this is some finite number of bits. Second, there is always a notion of time when we talk about state. Usually, we think of time being involved with state, because it may change over time.
Let’s think of an object, it starts off in an initial state, and over time, that state changes. For example, if the object is a piece of paper, when we are drawing we may draw or erase something (add or remove elements) over time. Our picture’s state may not keep track of all the things that have happened to it, nevertheless, the picture changes from state to state, as various operations are performing object.

Learn More

Back to top

Controller

Background

In our design, after having designed the flip flop, the device can remember the information of its state, the next step is to transform the state into an actual function. To achieve this transformation, we first needed a “reader” to read out the current state. At this point the control unit comes into play. A control unit is a DNA sequence with recombinase sites whose expression is controlled by recom-binase and RDF by reversing or deleting a promoter and/or a terminator. To make the control unit reliable and predictable, we first need to be able to predict the behaviors of its “building blocks” (or “elements” in electrical engineering), from which we weave our engineer’s perspective into the biolog-ical system. However, we need to make some adaptations and adjustments to these “elements” to make them usable.

Aim

To characterize a group of terminators and measure their strength in both orientations.
To select unidirectional terminators that are able to serve as switches in genetic circuits.
To measure the strength of terminators flanked by one set of attB/P sites, and the inversion efficiency of recombinases.
To measure the terminator strength of terminators flanked by two sets of attB/P sites, and the inversion efficiency of different recombinases.
To construct a 7-segment display which is able to count from 1 to 3.

Key Achievements

In our project, after we have made these constructive modifications to terminators and recombinases to meet our requirements of robust, reliable and predictable behaviors, which will be presented in the form of a seven-segment display. This will improve our quantitative understanding of natural phenomena and foster an engineering discipline for obtaining new complex cell behaviors.
We measured the strengths of a total of 24 terminators in both directions, and we elected 6 suitable unidirectional terminators.
We measured the strengths of several terminators paired with attB/P sites and the inversion efficiency of 3 recombinases.
We succeeded to construct the transformation of the pattern from ”2017” to ”PKU”.

Our Approach and Designs

Characterization of Terminators

Terminators are responsible for disengaging the RNA polymerase from the DNA and stopping gene transcription in vivo. This characteristic makes terminators desirable as switches in synthetic genetic circuits. The terminators that are used to realize this function should be unidirectional. That is, they should be able to block the movement of RNA polymerase effectively in one orientation and hardly influence its movement in the other. With this “reader”, advantages include a shorter sequence, per-manently recorded message in the DNA and greater number of design plans. Consequently, it is es-sential that we characterize the different terminators. We first chose 24 terminators whose forward orientation strength is high, as previously reported. We then measured their strength in both the forward and reverse orientations in our testing system. Finally, we sifted out a set of six reliable unidirectional terminators without potential cryptic promoters in both orientations.

Given that the same part can perform differently in different genetic circuits, previous testing systems and results of terminators may not fit our project. We therefore constructed a rather simple but practical reporter plasmid (as shown below), based on which terminator strength can be measured via fluorescence intensity and simple calculations.

Vector construction

Figure 1. Vector construction to measure the terminator strength

As shown above (Figure 1.), the circuit used to measure terminator strength was constructed on the pSB4C5 vector. The terminator to be tested is located downstream of the Ptac promoter and followed successively by RiboJ, RBS, and sfGFP. To enable high throughput, we introduced a pair of BsaI cutting sites between Ptac and RiboJ so that Golden Gate assembly can be used to add different terminators efficiently. In addition, there is a random sequence (from a previous associated research, the author of which had designed it using the Sequence Manipulation Suite) between the two Bsal sites flanked by two RiboJ, which was constructed as a positive control.
The terminators to be tested were selected from the literature.

RiboJ acts as an insulator between the RBS and its upstream sequence in the genetic circuit. The insulating function of RiboJ is directly associated with its biochemical function. RiboJ can be defined as the DNA sequence containing a ribozyme called sTRSV- ribozyme and a 23-nt hairpin. The downstream hairpin helps to expose the RBS. mRNA can be cleaved by sTRSV autocatalytically at a defined residue. As a result, extraneous RNA leaders that arise from transcription from promoters with different start sites will be cleaved. Moreover, to make sure the sequence upstream of the RBS will not affect RiboJ’s efficiency, the transcripts of our genetic circuits were designed to have the same 5’-UTR sequence, to avoid the effect called “part junction interference”, in which neighboring sequences may alter the part’s character. For example, a barcode part was found to contain a sequence similar to the –10 region of a constitutive promoter.