Team:Peking/Project
Introduction
Cells are responsive to a myriad signals under most conditions. 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 , 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. 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. 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.
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 . 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. In 2010, a bistable push-on push-off switch was constructed as a memory module by Lou et al. Five years later, Pakpoom Subsoontorn et al. designed a toggle flip-flop switch whose state can be changed by the same signal. Moreover, in 2012, Jerome Bonnet demon-strated a rewriteable recombinase-addressable data (RAD) module. 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.
These studies indicate that more functions could be accomplished with a memory module embedded within the circuit. 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.
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.
Background
Building blocks for constructing a page layout.
How to install MDL
How to install MDL
How to install MDL
How to install MDL
How to install MDL
How to install MDL
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Basic MDL Usage
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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) need linkWhen 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.
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.
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.
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.FORM
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.For more detailed measurements, the expression vector and reporter of a recombinase were used to co-transform E. coli Top10 and samples were prepared for flow cytometry reading. Single colonies were picked and used to inoculate 1ml of LB media with antibiotics in a V-bottom 96-well plate. The cultures were grown at 37℃ and 1000 RPM for 12h. Subsequently, an aliquot comprising 2μl 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℃ and 1000 RPM for 15h. An aliquot comprising 2 μL 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 subset and GFP subset. The recombination efficiency was estimated from the proportion of the RFP subset in the total fluorescent population.
Figure 6. Gating of the RFP and GFP subsets and change of fluorescence after induction. Left: no inducer. Right: 10 mM arabinose for 15h.
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. A. The polycistronic structure containing the integrase and its corresponding RDF. B. 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.
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 8. 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 9. 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 10. 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 11. 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
Fine-tuning of Recombination Efficiency
Selection of an appropriate replication origin and RBS
For the vector with ColE1 and p15A replication origin, we found proper RBS in a list of calculated RBSs for TP901-1 and Bxb1 gp35.
Figure 12. 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
Figure 13. Bxb1 gp35 recombination efficiency with a variety of calculated RBS and RBS sequences from iGEM (B0030∼B0035). T.I.R = Translation Initiation Rate
For the expression vector with p15A replication origin, a proper RBS for TP901-1 was selected.
Figure 14. 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 15. The transfer curves of the recombinases we utilized. A. The transfer curve of integrase phiC31. B. The transfer curve of integrase Bxb1.
It is necessary to know how long it takes for complete flipping, since the interval between two input signals must be longer than the flipping time to enable the bio-flip-flop to work properly.
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.
Measurement of the Flipping Efficiency of Excisionases
Figure 16. 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 17. 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 18. 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.
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.
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
The circuit used to measure terminator strength was constructed on the pSB4C5 vector. The termina-tor 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 de-signed 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.
Ribo J 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- ribozyme26 and a 23-nt hairpin. The downstream hairpin helps to expose the RBS19. 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 se-quence similar to the -10 region of a constitutive promoter.
Flow Cytometry
Vectors constructed with different terminators in both directions were tested via flow cytometry.need link
Terminator strength
As shown below, a mathematical formula was also used to characterize the strength of the termina-tors. "Ts" is the abbreviation of "terminator strength". [GFP]random sequence denotes the fluores-cence strength of the plasmid with the random sequence. [GFP]terminator denotes the fluorescence strength of the plasmid with a terminator.need equation
Ideal unidirectional terminators should fit the following standards:
- The Ts of both directions are larger than 0.9 (i.e. there is no cryptic promoter potential)
- The Ts ratios (Ts of the forward direction/Ts of the reverse direction) are larger than 10
- The larger Ts between the forward and reverse directions has to be larger than 10 (which ensures the blocking strength to be high enough)
- The smaller Ts between the forward and reverse directions has to be no larger than 1.2 (i.e. terminators in reverse orientation no longer block the transcription process).
Characterization of Inversion Efficiency
After finishing these measurements, the main protagonist of our project, the recombinase, will come into play. When terminators are flanked by a pair of attB/P sites, the synthetic circuit will transit between on and off state, which is achieved by controlling the expression of the respective recombinases. Here the question becomes whether the attB/P sites flanking the terminators may alter their strength due to some unknown interaction. Thus, we needed to remeasure their strength. Additionally, the inversion efficiency of respective recombinases need to be characterized.We introduced the testing system and expression system of the corresponding recombinases into the same cell to see if inversions happened, and if leaky expression of recombinases would impact the system. In the end, couples of terminators and recombinases with near-complete inversions and minimal leakage were selected.
Vector construction
As shown above, we selected terminators for both directions that were sandwiched between attB/attP recognition sites, on the basis of the aforementioned terminator strength testing system. The constructed terminators are shown in the table below.
need table
The expression system includes the recombinase Φc31, Bxb1, TP901-1, or (int2?) under the control of the inducible pBAD promoter and optimal RBS, which can simultaneously ensure high efficiency and low leakage.
Experimental Design
- Group 1 is the experimental group.
- Group 2 is the test group for leakage determination.
- Group 3 is the group with ideal initial conditions when a terminator in the forward direction is inversed, where GFP will not be expressed under induction by IPTG.
- Group 4 is the ideal terminal condition, where GFP will be expressed under the induction of IPTG.
- Groups 5&6 are used to double-measure the Ts of the terminators.
- Groups 7&8 are control groups.
- Comparisons between groups 3&5 and groups 4&6 are used to investigate the possible alteration of terminator strength due to the introduction of recognition sites.
Activation and induction
Escherichia coli colonies were picked and grown for 12h in LB media containing antibiotics in culture tubes at 37°C and 250 rpm in an incubator. Next, the bacterial cultures were diluted at a ratio of 1:100 with fresh M9 medium with antibiotics and different concentrations of inducers, after which the bacteria were grown for another 12h.A microplate reader was used to conduct preliminary measurements.
The bacterial culture was transferred into a 96-well plate (200µl per well), and OD600 and fluores-cence intensity were measured. Background OD600 and fluorescence of the plate, culture medium, and autofluorescence were eliminated using appropriate controls. The ratio of fluorescence intensi-ty/OD600 was calculated using the net fluorescence intensity and net OD600. The transcription barrier strength of an attB/P-Terminator can be characterized quantitatively using the Ts formula.
Flow cytometer
Double/triple transformationEscherichia coli strains were grown for 12h in LB medium containing antibiotics in culture tubes at 37°C and 250 rpm in an incubator. Next, the cultures were diluted at a ratio of 1:100 with fresh M9 medium with antibiotics and different concentrations of inducers. These bacterial cultures were grown for 12h, after which fluorescence was measured on a Varioskan© Flash.
The cells were analyzed by flow cytometry using a BD biosciences Fortessa flow cytometer with blue (488-nm) and red (640-nm) lasers. An injection volume of 15 μl and a flow rate of 1 μl/s were used. Cytometry data were analyzed using Flowjo. The gated populations consisted of at least 10 000 cells. The median fluorescence of the gated populations was calculated using FlowJo and used for all reporting. Autofluorescence of white cells was subtracted from all fluorescence measurements.
Standards for determining excellent combinations of attB/P+Terminator
The ideal attB/P+terminator combinations should fit the following standards:
- The “Ts “ in both directions is larger than 0.8 (no distinct cryptic promoter potential exists)
- The Ts ratios (Ts of the forward direction/Ts of the reverse direction) are larger than 10
- The larger Ts between the forward and reverse directions has to be larger than 10 (which ensures the blocking strength to be high enough)
- The smaller Ts between the forward and reverse directions has to be no larger than 1.2 (i.e. terminators in reverse orientation no longer block the transcription process).
Characterization of Terminators Flanked by Two Pairs of attB/P Sites
To achieve a more complex function, we need far more than just one recombinase and one pair of attB/P sites. We thus introduced two pairs of attB/P sites on both sides of the terminator. These two pairs of attB/P sites make it possible to conduct a double-inversion, whose efficiency is solely decided by the product of the efficiencies of the two recombinases. A lower efficiency may also lead to an accumulation of errors, and once the accumulative effect is large enough, the entire logic circuit will deviate or even become blocked. To circumvent this problem, we needed to make sure that the recombinases we selected have high efficiencies to invert the sequence between the attB/P sites, not once, but twice. The inversion efficiencies of different recombinases are shown on this page (click here). need linkVector Construction
As shown above, the terminator is sandwiched between two different pairs of attB/attP sites, under the control of a constitutive J23119 promoter.
We built the following constructs:
need table
Two sets of expression systems, induced by IPTG and arabinose, were constructed.
Flow Cytometry
Construction of a Seven-Segment Display
After constructing two reliable elements with predictable behaviors, we aimed to directly implement and arrange them according to sequential logic, which comes in the form of a visible “1→2→3” transition on the seven-segment display. In this display, GFP expression is used instead of an LCD (liquid crystal display) to light up the panel. In our case, three simple and previously characterized parts were placed in segments except F. The system yields the predictable expression of recombinases which invert terminators between cor-responding attB/attP sites either once (A,D,G) or twice (C,E).
Results
Characterization of Terminators
A total of 24 terminators were tested in both orientations (their code names[1] and abbreviations are shown in the table below). And a gradient of different concentrations of IPTG (0, 0.01, 0.1, 1mM) was used for induction. We found that the calculated Ts values varied with IPTG concentration. Commonly, the Ts value for the forward orientation increased with IPTG concentration and reached the highest value at 0.1mM, after which it declined slightly. In the reverse orientation there were no obvious trends. Consequently, 0.1mM of IPTG was the most suitable concentration for induction. After the test, we chose 6 terminators (221,836,435, 322,855 and 309), among which 435 and 322 showed the best performance and conformed best to the 4 standards described above.need table
(The code names and their abbreviations.)
Characterization of Inversion Efficiency
Ts of the different attB/P+terminator combinationsTransformation with the testing system plasmid alone yielded the following results and conclusions:
Sole attB/P sites (without a terminator) can block the transcription to a certain extent. Such barrier strength varied with the types of the sites. Among the 5 pairs of attB/P sites tested, int2 had the strongest intrinsic barrier effect ("Ts"= 23.0), even exceeding that of the terminator 221F ("Ts"=16.6). The other sites had weaker barrier effects: int2＞ΦC31 ("Ts"=2.00)＞TP901-1(AG)("Ts"=1.42).
Combination of different attB/P sites and terminators can produce unexpected results
Int2 attB/P and 435F had the strongest barrier strengths among the recognition sites and terminators, respectively. However, the barrier strength of their combination was the weakest (“Ts”=5.37) among all combinations.TP901-1(AG), attB/P and 221F had the weakest barrier strength among recognition sites and terminators, respectively, but the barrier strength of their combination was the highest.
In addition to the barrier effect, the combination of attB/P sites and terminators can also lead to the formation of cryptic promoters, which was the case for the TP901-1(AG) 221R combination.
The most excellent combinations of attB/P sites and terminators selected based on the four standards above were (with the corresponding “Ts” ratios in brackets): int2 221 (35.3), Φc31 435 (28.3), TPAG 221 (17.7), TPAG 435 (17.3), int2 855 (17.2), and BxGT435 (21.1).
Considering that the expression systems of int2 and TP901-1 are induced by IPTG, which causes a conflict with the reporting system, we used BxGT435 and Φc31 435, whose systems are induced by Ara, to conduct the reversal experiments.
Reversal Experiment
Co-transformation of E. coli with the testing system plasmid and corresponding recombinase expression system We observed successful reversal events after inducing the expression of the recombinase. As shown in the picture of the 96-well plate, the fluorescence difference is visible by naked eye. Using the microplate reader and analysis, we confirmed the existence of a significant fluorescence difference. In addition, we directly used the bacterial solution to conduct a PCR, and the sequencing results showed double peaks, which substantially proves that the reversal occurred.
Characterization of Terminators Flanked by Two Pairs of attB/P Sites
We built the following constructs:need table
Two sets of expression systems, induced by IPTG and arabinose, were constructed.
Ts of double attB/P+terminator combinations
Due to the introduction of double attB/P sites, which substantially extended the distance from the promoter to the GFP coding sequence, together with the barrier effect of each pair of attB/P sites, it became impractical to expect the Ts of the attB/P with a reverse terminator to be around 1. We there-fore lowered the standard and laid more emphasis on the ratio of Ts for both orientations. Based on the test results above, we chose TP901-1 BxGT435 and Φc31 BxbGT 435 to conduct the reversal experiment.
Reversal Experiment
Construction of the Seven-Segment Display
need pictureReferences
- Dyakonov T, Muir A, Nasri H, et al. Characterization of 582 natural and synthetic terminators and quantification of their design constraints[J]. Nature Methods, 2013, 10(7):659-664.
- Brophy J A, Voigt C A. Antisense transcription as a tool to tune gene expression[J]. Molecular Systems Biology, 2016, 12(1):854.
- Yang L, Voigt CA, et al. Permanent genetic memory with >1-byte capacity. Nat Methods. 2014 Dec; 11(12):1261-6.
- Stothard P. The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques. 2000 Jun; 28(6):1102, 1104.
- Lou C, Stanton B, Chen YJ, Munsky B, Voigt CA. Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nat Biotechnol. 2012 Nov; 30(11):1137-42. [PMID:23034349]
Clock
Background
Clock Signal
A clock signal is a particular type of signal that oscillates between a high and a low state repeatedly. This signal is emitted by a clock generator and is utilized like a metronome to trigger actions of a sequential logic circuit. In one clock signal cycle, the state of the circuit, which is the basis of sequential logic circuits, remains unchanged after shifting. A sequential logic circuit designed with an inner clock can change its state automatically, even without inputs. Additionally, the time a state lasts can be controlled by the clock signal period.
Figure 1. Typical clock signal and fluorescence signals of the repressilator.
Repressilator Introduction
The repressilator is a synthetic genetic regulatory network consisting of a ring-oscillator with three genes -- tetR from the Tn10 transposon, cI from bacteriophage λ and lacI from the E. coli lactose operon, each expressing a protein that represses the next gene in the loop.
Figure 2. The structure of the original repressilator. It is encoded by a low-copy plasmid with three repressor genes and a high-copy plasmid containing a GFP gene driven by PLtetO1.
The Repressilator as an Internal Clock
In our project, we aimed to use the repressilator to generate a clock signal. We chose it for the following reasons:- It can generate up to three oscillatory signals in different phases; two different signal peaks can drive the flip-flop to shift its state serving as a component of a period.
- The period and amplitude of the oscillation can stay stable and robust through many generations.
- The period of the oscillation is adjustable. We can modify the oscillation period by simply chang-ing the bacterial generation period and oscillator components.
- It has the potential to be connected to a downstream circuit. Because of the oscillatory changes in promoter strength, we can add the promoters (PR ,PLtetO1 ,PLlacO1) to the downstream circuit.
Aims
Compared with natural biological systems, synthetic circuits such as the repressilator have a lower accuracy of oscillation. There have been many efforts to enhance the robustness of oscillation of the original repressilator, such as improvements in the core cellular processes and the reporter systems . Thus, if we want to use the repressilator as an internal clock, we have to determine whether our construct would have a significant influence on the oscillation itself. To answer this question, we have to do the following experiments:- Evaluate the repressilator system in our own experimental environment.
- Construct an oscillator-induced protein expression system and compare its oscillation period with the parent repressilator.
Key Achievements
- Test the parent repressilator published in the 2016 paper by Potvin-Trottier et al.
- Construct a recombinase oscillator and T7 oscillator.
Our Approach
Reproduction of the Oscillation of the Original Repressilator
We used a stereoscope, a microfluidic device and a plate reader to reproduce the oscillation experi-ments of the parent repressilator (single reporter oscillator and three-reporter oscillator). The main strains we used in the oscillation-related experiments are listed in Table 1. The clpXP genes in these strains were deleted to obtain more robust oscillations.Table1. The strains used in the oscillation-related experiments.
| Strain | Plasmid | Genotype (MC4100) |
|---|---|---|
| Recombinase Oscillator | plpt20-gp35 plpr41-reporter |
ΔclpXP |
| Recombinase Amplifier | plpt20-t7 plpt41-gp35-reporter |
ΔclpXP |
| T7 Polymerase Oscillator | plpt20-t7 plpt41-t7 test |
ΔclpXP |
| Triple Reporter Oscillator (LPT117) | plpt107 plpt43 |
ΔclpXP |
| Single Reporter Oscillator | plpt20 Plpt41 |
ΔclpXP |
Since one of the repressors is under the control of PLlacO1, we can use IPTG (Isopropyl β-D-Thiogalactoside) to synchronize the oscillation in different cells. We also compared the synchronized oscillation and the unsynchronized oscillation. (see Results figure)
If we want to look at dynamic changes of the oscillation, population-based experiments are not enough, so we also designed a microfluidic device of the same type as the "mother machine" used in our lab to house single cells and their offspring. In this device, cells are trapped in narrow channels and newborn cells are washed away by fresh medium. (see Results video1)
need picture
Figure 3. Design of the "mother machine". It consists of dozens of thin channels holding single cells, and can be used to track individual cell line for generations.
Design of the Recombinase Oscillator
In order to test whether our modifications of the repressilator would severely affect its oscillation pattern, we combined the repressilator with recombinases (Bxb1 gp35) to obtain an oscillator-activated recombinase expression system. By adjusting this system, we can also determine whether the oscillation period is compatible with the recombination time.
Figure 4. Schematic diagram of the recombinase oscillator.
For the expression system of the recombinase oscillator, we made use of the plpt20 plasmid (Laurent et.al., 2016), and we cloned Bxb1 gp35 downstream of mVenus in plpt20. By doing this, the expression of recombinase was coupled to the expression of mVenus, which is under the control of the promoter PLtetO1.
As for the reporter system used to detect recombinase expression, we constructed a revertible promoter surrounded by a Bxb1 attB site and a Bxb1 attP site on the sponge plasmid. When the recombinase is expressed, it inverts the promoter sequence and mRFP is expressed.
Both the expression and reporter systems of the recombinase oscillator were constructed using Gibson assembly.
A
B
Figure 5. Plasmids of the recombinase oscillator. (A) Plasmid encoding the oscillatory recombinase expression system (plpt20-gp35). (B) The plasmid encoding the oscillatory recombinase reporter system (plpt41-reporter).
Possible improvements
- mRFP is a widely used fluorescent protein in mammalian cells, but it has its drawbacks. As we have discovered in our experiments, mRFP takes a rather long time to mature and emit red fluorescence. Thus, we may have to exchange the fluorescent reporter protein for a fast-maturing one such as mKate.
- The oscillation period is not compatible with the duration of recombinase-induced promoter reversal and mRFP maturation. In order to adjust the oscillation period, we can use different liquid media to prolong the oscillation period. Previous work has shown that the oscillation period is proportional to the cell division time. Thus, by using a liquid medium such as M9, we can increase the cell division time and prolong the oscillation period.
Design of the T7-coupled Oscillator
The repressilator is constantly working as an internal clock, but under most circumstances we would like to start the internal clock in a specific moment. Using IPTG to synchronize the internal clocks of all the cells in a population and keep them in the same phase seems plausible. However, through our experiments with the recombinase oscillator we found that using IPTG cannot prevent signal leakage of the internal clock. Thus, we would like to add an "AND" gate behind the clock signal, so that we can turn on or turn off the internal clock.T7 RNA polymerase has been previously used to implement an "AND" gate. In that design, the first promoter controls the expression of a T7 RNA polymerase gene with two internal amber stop codons. The second promoter controls the amber suppressor tRNA supD. When both promoters are activated, T7 RNA polymerase is expressed and can act specifically on the downstream T7 promoter. What's more, T7 RNA polymerase can be used as an amplifier to increase the signal amplitude.
Our aim is to prove that T7 RNA polymerase can be combined with the repressilator and can serve as a potential on-off switch and amplifier. Therefore, we designed a T7 oscillator and recombinase amplifier.
Figure6. Schematic diagram of the recombinase oscillator.
A
B
C
Figure 7. Plasmids of the T7 polymerase oscillator. (A) The plasmid encoding the T7 oscillator expression system (plpt20-T7). (B) The plasmid encoding the T7 oscillator reporter system (plpt41-T7 test). (C) The plasmid encoding the recombinase amplifier system.
For the expression system of the T7 oscillator, we also used the plpt20 plasmid (Laurent et.al. , 2016). We cloned the T7 RNA polymerase right behind mVenus in plpt20. To detect the expression of T7 RNA polymerase, we added a T7 promoter before mRFP in the plpt41 plasmid. In order to prove that T7 polymerase can be used as an amplifier, we also constructed a recombinase amplifier. The re-porter system of the recombinase amplifier was adapted from the plpt41-reporter; we added a Bxb1-gp35 activated by a T7 promoter in the plpt41-reporter plasmid.
Microfluidic Image Analysis
Firstly, we opened the files from the microscope generated in nd2 format in NIS element viewer or image J plugins NDi6d. We were then able to change them to videos or pictures in Tag Image File Format. Finally, we used image J to analyze the intensity of the images in TIF format.Results
Recurrence of the Oscillation in the Repressilator
need pictureFigure 8. Stereoscopic image of colony oscillation. Triple reporter Plpt107+titration sponge. We repeated the experiments of the original repressilator (John et al., 2016,) using the LPT117 strain. By using a stereoscope, we discovered concentric yellow fluorescence circles in the colony of LPT117. This spatial pattern is the reflection of temporal pattern of the oscillation. It is formed when the fluorescence signal oscillates during cell division, and inner parts of cells in the colony cannot divide and remain the fluorescence signal.
We repeated the experiments of the original repressilator (John et al., 2016,) using the LPT117 strain. By using a stereoscope, we discovered concentric green fluorescence circles in the colony of LPT117.
A
B
Figure 9. Typical time trace of a single cell for original repressilator. A. Time trace of LPT117 repressilator (plpt107+titration sponge). B. Time trace of single reporter repressilator (plpt20+titration sponge). Both traces were normalized to their means. The oscillation period of LPT117 is around 6 hours in LB medium. The oscillation period of the single reporter oscillator is also around 6 hours.
need video
Video 1. Microfluidic videos of oscillations in the mother machine. A. LPT117 repressilator. B. single reporter repressilator.
Recombinase Oscillator
Figure 10. Optical microscopic views of a bacterial colony with a “disk-like” pattern. An optical microscopic view of a bacterial colony with a “disk-like” pattern, the red parts of the colony are distributed in wedge form. When recombinase Bxb1-gp35 expresses, the promoter of mRFP surrounded by Bxb1 attB and attP sites is inverted, which leads to the expression of mRFP. Some cells irreversibly shift from red to light yellow owing to loss of the separate mRFP-expressing reporter plasmid.
Figure 11. Time trace of a single cell for recombinase oscillator. The oscillation period of recombinase oscillator is around 6 hours in LB medium, which is similar to the oscillation period of LPT117 and single reporter repressilator. These results shows that our modification on the parent repressilator (combine recombinase with repressilator) will not affect the clock itself and clock signal can be passed from the repressilator to downstream structure (invert of mRFP promoter).
Video 2. Microfluidic video of Recombinase Oscillator.
T7 Polymerase Oscillator and Recombinase Amplifier
need pictureFigure 12. Proof of the reliability of the reporter systems in the T7 polymerase oscillator and recombinase amplifier.
References
- Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Na-ture, 403(6767), 335-338.
- Potvin-Trottier, L., Lord, N. D., Vinnicombe, G., & Paulsson, J. (2016). Synchronous long-term oscilla-tions in a synthetic gene circuit. Nature, 538(7626), 514-517.
- Wang, P., Robert, L., Pelletier, J., Dang, W., Taddei, F., Wright, A., & Jun, S. (2010). Robust Growth of Escherichia coli. Current Biology, 20(12), 1099–1103.
Demostrate
Background & Design
We attempts to develop a framework 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.Methods
The bio-flip-flop is constructed as two sequences each with an invertible promoter flanked between recombination sites and expressing recombinase or recombinase-RDF fusion protein mediating inversion of another sequence.need picture
Fig.1. Bio-flip-flop construction.
We first tested and optimized recombinase and recombinase-RDF fusion protein functions and we standardized these parts into BioBrick devices. Then we separated the function of bio-flip-flops and built two circuits: the Forward and Backward latch. Each of them can implement state change when receives a group of trigger signal consisting of two inducing events. We then introduced these latches in vivo and proved their functionality.
link to flip-flop
need picture (Forward latch and Backward latch)
Fig.2. Construction of Forward and Backward latches of the bio-flip-flop and their function.
need data
Unidirectional terminators whose directions are changed by recombinases or recombinase-RDF fusion proteins are used to control transcription of the target gene. We measured the strengths of 24 terminators in both directions, and we elected six suitable unidirectional terminators. We measured the strengths of several terminators paired with attB/P sites and the inversion efficiency of three recombinases.
need data
link to terminator
We used repressilator, a classic genetic oscillator, as an internal clock to drive our bio-flip-flop. To do so, we first observed and evaluated the repressilator system in our own experimental environment. We then constructed an oscillator-induced recombinase expression system and observed promoter inversion, which shows that repressilator has the possibility to act as an internal oscillatory trigger signal generator.
link to oscillator
Results
Following the methodology of modularity in synthetic biology, we divided the system into three key components. We showed the feasibility of their assembly into a whole system by a set of simulation results.First, an ordinary differential equation system describing recombinase and RDF function was built. Parameters were estimated from experimental data. Then we assembled four components (two recombinases and two fusion proteins) as the one in flip-flop design. Inducing of the system is in the form of square waves to simulate manual induction like changing of medium.
need two picture
Fig.3. Simulation of the whole flip-flop function. X_F denotes the fraction of forward direction (right) of the upper promoter pBAD. Y_F denotes the fraction of forward direction (right) of the lower promoter pTAC.
We also proved that the system can also be triggered by repressilator as internal clock signal. Substituting the upper promoter (repressed by tetR protein) to PLtetO1 and the lower one (repressed by cI protein) to PR, we then did another simulation of forward latch and proved that the flip-flop can change its state and then recover back to initial state after arrival of two adjacent trigger signal groups.
need two picture
Discussion
In this simulation experiment, the flip-flop lost approximately 10% of its initial state after two trigger signal groups. If we use manual and discrete induction, the loss is roughly 5%. These results have reached the expectations. However, in practical experiment, there are still many problems to solve and further research is needed. Though we constructed and configured a set of recombinase and recombinase-RDF fusion protein tools, we observed leaky expression and other problems when assembled. Further modification of repressilator (adding one more recombinase) has to be tested to satisfy the need of in vivo automatic state transition.Potential Applications
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. We also illustrated the possibility of self-triggered state transition. Possible application would be an automatic cell factory that can complete a series of manufacturing procedures with just one trigger at the start. Furthermore, our bio-flip-flop can act as an in vivo memory device and aid in recording cellular events, which can provide deep insight into cell differentiation and development.Background
Building blocks for constructing a page layout.
How to install MDL
How to install MDL
How to install MDL
How to install MDL
How to install MDL
How to install MDL
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Basic MDL Usage
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