Difference between revisions of "Team:Peking/Project"
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<section id="Flip-flop" class="mdl-components__page mdl-grid"> | <section id="Flip-flop" class="mdl-components__page mdl-grid"> | ||
<div class="demo-card-wide mdl-card mdl-shadow--2dp"> | <div class="demo-card-wide mdl-card mdl-shadow--2dp"> | ||
| − | + | <h1>Flip-flop</h1> | |
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| + | <div class="mdl-card__supporting-text"> | ||
<section class="docs-toc docs-text-styling" style="margin-left: 0px"> | <section class="docs-toc docs-text-styling" style="margin-left: 0px"> | ||
<nav class="section-content" style="margin-left: 0px"> | <nav class="section-content" style="margin-left: 0px"> | ||
<ul style="margin-left: 0px"> | <ul style="margin-left: 0px"> | ||
| − | <li><a href="# | + | <li><a href="#p1">Background</a></li> |
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| + | <li><a href="#p2">Description</a></li> | ||
| + | |||
| + | <li><a href="#p3">Key Achievements</a></li> | ||
| + | <li><a href="#p4">Our Approach and Designs</a></li> | ||
| + | <li><a href="#p4.1" style="padding-left: 20px;font-size: small">Design of the bio-flip-flop</a></li> | ||
| + | <li><a href="#p4.2"style="padding-left: 20px;font-size: small">Selection of available integrases</a></li> | ||
| + | <li><a href="#p5">Standard Characterization & Optimization Workflow</a></li> | ||
| + | <li><a href="#p5.1"style="padding-left: 20px;font-size: small">Measurement of recombination efficiency</a></li> | ||
| + | <li><a href="#p5.2"style="padding-left: 20px;font-size: small">Characterization of excisionases</a></li> | ||
| + | <li><a href="#p6">Construction of bio-flip-flops</a></li> | ||
| + | <li><a href="#p6.1"style="padding-left: 20px;font-size: small">Forward latch</a></li> | ||
| + | <li><a href="#p6.2"style="padding-left: 20px;font-size: small">Backward latch</a></li> | ||
| + | <li><a href="#p6.3"style="padding-left: 20px;font-size: small">Experiment</a></li> | ||
| + | <li><a href="#p7">Results</a></li> | ||
| + | <li><a href="#p7.1"style="padding-left: 20px;font-size: small">Fine-tuning of recombination efficiency</a></li> | ||
| + | <li><a href="#p8">References</a></li> | ||
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</ul> | </ul> | ||
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</section> | </section> | ||
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| − | + | <h2 id="p1">Background</h2> | |
| − | + | 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)<br><br> | |
| − | + | When designing genetic-circuit counterparts of flip-flops, we should consider the following three criteria:<br> | |
| − | + | <ul> | |
| − | + | <li>The device should have at least two stable states;</li> | |
| − | + | <li>State changes are triggered by an identical signal;</li> | |
| − | + | <li>The flip-flop should hold its state and remain unaltered until the next signal arrives.</li> | |
| − | + | </ul> | |
| − | + | 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.<br><br> | |
| − | + | 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.<br><br> | |
| − | + | ||
| − | + | ||
| − | + | Fig1. Site-specific recombination either integrates, deletes or reverses a DNA sequence.<br><br> | |
| − | + | 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.<br><br> | |
| − | + | 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.<br> | |
| − | + | Fig2. Schematic drawing of the RDF mechanism. (Olorunniji et al. 2017)<br> | |
| − | + | ||
| − | + | <h2 id="p2">Aims</h2> | |
| − | + | 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:<br> | |
| − | + | <ul> | |
| − | <br><br> | + | <li>Select effective recombinases and their corresponding RDFs; characterize and optimize their function.</li> |
| + | <li>Construct half of the flip-flop structure and express it in <i>E. coli.</i></li> | ||
| + | </ul> | ||
| + | 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.<br><br> | ||
| + | This would serve as a proof of concept for a novel design strategy for bio-flip-flops.<br> | ||
| + | |||
| + | <h2 id="p3">Key Achievements</h2> | ||
| + | <ul> | ||
| + | <li>Test and optimize recombinase- and recombinase-RDF fusion protein function.</li> | ||
| + | <li>Standardization into BioBrick devices.</li> | ||
| + | <li>Introduction of the forward latch of the bio-flip-flop into cells and proof of function in vivo.</li> | ||
| + | </ul> | ||
| + | |||
| + | <h2 id="p4">Our Approach and Designs</h2> | ||
| + | <h3 id="p4.1">Design of the bio-flip-flop</h3> | ||
| + | Using a pair of integrases and their corresponding excisionases, we designed the bio-flip-flop device to store state information as follows:<br> | ||
| + | <ul> | ||
| + | <li>The device has two promoters, controlled by input inducers X and Y, respectively. The input can either promote or inhibit transcription.</li> | ||
| + | <li>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.</li> | ||
| + | <li>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.</li> | ||
| + | </ul> | ||
| + | |||
| + | Fig3. Schematic diagram of the bio-flip-flop mechanism.<br> | ||
| + | |||
| + | <h3 id="p4.2">Selection of available integrases</h3> | ||
| + | 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.<br><br> | ||
| + | FORM<br><br> | ||
| + | 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.<br> | ||
| + | |||
| + | <h2 id="p5">Standard Characterization & Optimization Workflow</h2> | ||
| + | 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.<br><br> | ||
| + | |||
| + | Figure 4. The workflow of bio-flip-flop optimization.<br><br> | ||
| + | |||
| + | 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 <i>E. coli</i> Top10 with the expression vector and a reporter plasmid. The reporter plasmid expresses different fluorescent proteins before and after flipping of a constitutive promoter.<br><br> | ||
| + | |||
| + | Figure 5. The standard genetic structure used to characterize the recombinases.<br><br> | ||
| + | |||
| + | 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, <i>araC</i>, and an arabinose-induced pBad promoter. RiboJ was added to Plasmid2 by cloning RiboJ and the recombinase from Plasmid1, and assembled after pBad.<br><br> | ||
| + | |||
| + | Fig6. 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.<br><br> | ||
| + | |||
| + | 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.<br><br> | ||
| + | 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.<br><br> | ||
| + | |||
| + | <h3 id="p5.1">Measurement of recombination efficiency</h3> | ||
| + | We used a microplate reader to roughly measure the efficiency of the selected integrases. We used flow cytometry to conduct a more accurate characterization.<br><br> | ||
| + | For more detailed measurements, the expression vector and reporter of a recombinase were used to co-transform <i>E. coli</i> 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.<br><br> | ||
| + | |||
| + | Fig7. Gating of the RFP and GFP subsets and change of fluorescence after induction. Left: no inducer. Right: 10 mM arabinose for 15h.<br><br> | ||
| + | |||
| + | 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.<br><br> | ||
| + | |||
| + | <h3 id="p5.2">Characterization of excisionases</h3> | ||
| + | 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.<br><br> | ||
| + | |||
| + | Figure 8. 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 inte-grase with its corresponding RDF.<br><br> | ||
| + | |||
| + | The method is similar to the integrase test method. We constructed an input plasmid to express inte-grases 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.<br><br> | ||
| + | <h2 id="p6">Construction of bio-flip-flops</h2> | ||
| + | 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 process 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.<br><br> | ||
| + | <h3 id="p6.1">Forward latch</h3> | ||
| + | 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 irre-versible states (Figure 10).<br><br> | ||
| + | Fig9. 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.<br><br> | ||
| + | Fig10. 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.<br><br> | ||
| + | <h3 id="p6.2">Backward latch</h3> | ||
| + | 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.<br><br> | ||
| + | Fig11. The structure of the Backward latch. Two plasmids constitute the Backward latch. Each plas-mid 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.<br><br> | ||
| + | Fig12. 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.<br><br> | ||
| + | <h3 id="p6.3">Experiment</h3> | ||
| + | Because our final goal is to realize the state transition of our Bio-Flip-Flop, it is significant to charac-terize 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.<br><br> | ||
| + | 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.<br><br> | ||
| + | <h2 id="p7">Results</h2> | ||
| + | <h3 id="p7.1">Fine-tuning of recombination efficiency</h3> | ||
| + | <h4>Selection of an appropriate replication origin and RBS</h4> | ||
| + | For the vector with ColE1 replication origin, we found proper RBS in a list of calculated RBSs for TP901-1 and Bxb1 gp35.<br><br> | ||
| + | Fig13. TP901-1 recombination efficiency with a variety of calculated RBS. T.I.R = Translation Initiation Rate<br><br> | ||
| + | Fig14. Bxb1 gp35 recombination efficiency with a variety of calculated RBS and RBS sequences from iGEM (B0030∼B0035). T.I.R = Translation Initiation Rate<br><br> | ||
| + | For the expression vector with p15A replication origin, a proper RBS for TP901-1 was selected.<br><br> | ||
| + | Figure 15. TP901-1 recombination efficiency with a variety of RBS from iGEM (B0030∼B0035). T.I.R = Translation Initiation Rate<br><br> | ||
| + | <h4>Transfer curves and temporal curves</h4> | ||
| + | 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 con-centration.<br><br> | ||
| + | Figure 16. The transfer curves of the recombinases we utilized. A. The transfer curve of integrase phiC31. B. The transfer curve of integrase Bxb1.<br><br> | ||
| + | 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.<br><br> | ||
| + | <h2 id="p8">Reference</h2> | ||
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</div> | </div> | ||
Revision as of 16:33, 28 October 2017
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Background
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How to install MDL
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Tempor tempor aliqua in commodo cillum Lorem
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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)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.
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.
Fig1. Site-specific recombination either integrates, deletes or reverses a DNA sequence.
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.
Fig2. Schematic drawing of the RDF mechanism. (Olorunniji et al. 2017)
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 4. 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 different fluorescent proteins before and after flipping of a constitutive promoter.
Figure 5. 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.
Fig6. 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.
Fig7. 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 8. 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 inte-grase with its corresponding RDF.
The method is similar to the integrase test method. We constructed an input plasmid to express inte-grases 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 process 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 irre-versible states (Figure 10).Fig9. 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.
Fig10. 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.Fig11. The structure of the Backward latch. Two plasmids constitute the Backward latch. Each plas-mid 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.
Fig12. 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 charac-terize 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 replication origin, we found proper RBS in a list of calculated RBSs for TP901-1 and Bxb1 gp35.Fig13. TP901-1 recombination efficiency with a variety of calculated RBS. T.I.R = Translation Initiation Rate
Fig14. 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 15. TP901-1 recombination efficiency with a variety of RBS from iGEM (B0030∼B0035). T.I.R = Translation Initiation Rate
Transfer curves and temporal 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 con-centration.Figure 16. 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.
Reference
Background
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incididunt mollit duis dolore amet amet tempor exercitation. Qui amet aute ea aute id ad aliquip proident. Irure duis qui labore deserunt enim in quis nisi sint consequat aliqua. Ex proident labore et laborum tempor fugiat sint magna veniam minim. Nulla dolor labore adipisicing in enim mollit laboris fugiat eu. Aliquip minim cillum ullamco voluptate non dolore non ex duis fugiat duis ad. Deserunt cillum ad et nisi amet non voluptate culpa qui do. Labore ullamco et minim proident est laborum mollit ad labore deserunt ut irure dolore. Reprehenderit ad ad irure ut irure qui est eu velit eu excepteur adipisicing culpa. Laborum cupidatat ullamco eu duis anim reprehenderit proident aute ad consectetur eiusmod.
Background
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How to install MDL
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How to install MDL
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Basic MDL Usage
Cillum dolor esse sit incididunt velit eiusmod magna ad nostrud officia aute dolor dolor. Magna esse ullamco pariatur adipisicing consectetur eu commodo officia. Ex cillum consequat mollit minim elit est deserunt occaecat nisi amet. Quis aliqua nostrud Lorem occaecat sunt. Eiusmod quis amet ullamco aliquip dolore ut incididunt duis adipisicing. Elit consequat nisi eiusmod aute ipsum sunt veniam do est. Occaecat mollit aliquip ut proident consectetur amet ex dolore consectetur aliqua elit. Commodo nisi non consectetur voluptate incididunt mollit duis dolore amet amet tempor exercitation. Qui amet aute ea aute id ad aliquip proident. Irure duis qui labore deserunt enim in quis nisi sint consequat aliqua. Ex proident labore et laborum tempor fugiat sint magna veniam minim. Nulla dolor labore adipisicing in enim mollit laboris fugiat eu. Aliquip minim cillum ullamco voluptate non dolore non ex duis fugiat duis ad. Deserunt cillum ad et nisi amet non voluptate culpa qui do. Labore ullamco et minim proident est laborum mollit ad labore deserunt ut irure dolore. Reprehenderit ad ad irure ut irure qui est eu velit eu excepteur adipisicing culpa. Laborum cupidatat ullamco eu duis anim reprehenderit proident aute ad consectetur eiusmod. Cillum dolor esse sit incididunt veliteiusmod magna ad nostrud officia aute dolor dolor. Magna esse ullamco pariatur adipisicing consectetur eu commodo officia. Ex cillum consequat mollit minim elit est deserunt occaecat nisi amet. Quis aliqua nostrud Lorem occaecat sunt. Eiusmod quis amet ullamco aliquip dolore ut incididunt duis adipisicing. Elit consequat nisi eiusmod aute ipsum sunt veniam do est. Occaecat mollit aliquip ut proident consectetur amet ex dolore consectetur aliqua elit. Commodo nisi non consectetur voluptate incididunt mollit duis dolore amet amet tempor exercitation. Qui amet aute ea aute id ad aliquip proident. Irure duis qui labore deserunt enim in quis nisi sint consequat aliqua. Ex proident labore et laborum tempor fugiat sint magna veniam minim. Nulla dolor labore adipisicing in enim mollit laboris fugiat eu. Aliquip minim cillum ullamco voluptate non dolore non ex duis fugiat duis ad. Deserunt cillum ad et nisi amet non voluptate culpa qui do. Labore ullamco et minim proident est laborum mollit ad labore deserunt ut irure dolore. Reprehenderit ad ad irure ut irure qui est eu velit eu excepteur adipisicing culpa. Laborum cupidatat ullamco eu duis anim reprehenderit proident aute ad consectetur eiusmod. Cillum dolor esse sit incididunt velit eiusmod magna ad nostrud officia aute dolor dolor. Magna esse ullamco pariatur adipisicing consectetur eu commodo officia. Ex cillum consequat mollit minim elit est deserunt occaecat nisi amet. Quis aliqua nostrud Lorem occaecat sunt. Eiusmod quis amet ullamco aliquip dolore ut incididunt duis adipisicing. Elit consequat nisi eiusmod aute ipsum sunt veniam do est. Occaecat mollit aliquip ut proident consectetur amet ex dolore consectetur aliqua elit. Commodo nisi non consectetur voluptate
incididunt mollit duis dolore amet amet tempor exercitation. Qui amet aute ea aute id ad aliquip proident. Irure duis qui labore deserunt enim in quis nisi sint consequat aliqua. Ex proident labore et laborum tempor fugiat sint magna veniam minim. Nulla dolor labore adipisicing in enim mollit laboris fugiat eu. Aliquip minim cillum ullamco voluptate non dolore non ex duis fugiat duis ad. Deserunt cillum ad et nisi amet non voluptate culpa qui do. Labore ullamco et minim proident est laborum mollit ad labore deserunt ut irure dolore. Reprehenderit ad ad irure ut irure qui est eu velit eu excepteur adipisicing culpa. Laborum cupidatat ullamco eu duis anim reprehenderit proident aute ad consectetur eiusmod.
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
Tempor tempor aliqua in commodo cillum Lorem
magna dolore proident Lorem. Esse ad consequat est excepteur irure eu irure quis aliqua qui. Do mollit esse veniam excepteur ut veniam anim minim dolore sit commodo consequat duis commodo. Sunt dolor reprehenderit ipsum minim eiusmod eu consectetur anim excepteur eiusmod. Duis excepteur anim dolor sit enim veniam deserunt anim adipisicing Lorem elit. Cillum sunt do consequat elit laboris nisi consectetur.
Basic MDL Usage
Cillum dolor esse sit incididunt velit eiusmod magna ad nostrud officia aute dolor dolor. Magna esse ullamco pariatur adipisicing consectetur eu commodo officia. Ex cillum consequat mollit minim elit est deserunt occaecat nisi amet. Quis aliqua nostrud Lorem occaecat sunt. Eiusmod quis amet ullamco aliquip dolore ut incididunt duis adipisicing. Elit consequat nisi eiusmod aute ipsum sunt veniam do est. Occaecat mollit aliquip ut proident consectetur amet ex dolore consectetur aliqua elit. Commodo nisi non consectetur voluptate incididunt mollit duis dolore amet amet tempor exercitation. Qui amet aute ea aute id ad aliquip proident. Irure duis qui labore deserunt enim in quis nisi sint consequat aliqua. Ex proident labore et laborum tempor fugiat sint magna veniam minim. Nulla dolor labore adipisicing in enim mollit laboris fugiat eu. Aliquip minim cillum ullamco voluptate non dolore non ex duis fugiat duis ad. Deserunt cillum ad et nisi amet non voluptate culpa qui do. Labore ullamco et minim proident est laborum mollit ad labore deserunt ut irure dolore. Reprehenderit ad ad irure ut irure qui est eu velit eu excepteur adipisicing culpa. Laborum cupidatat ullamco eu duis anim reprehenderit proident aute ad consectetur eiusmod. Cillum dolor esse sit incididunt veliteiusmod magna ad nostrud officia aute dolor dolor. Magna esse ullamco pariatur adipisicing consectetur eu commodo officia. Ex cillum consequat mollit minim elit est deserunt occaecat nisi amet. Quis aliqua nostrud Lorem occaecat sunt. Eiusmod quis amet ullamco aliquip dolore ut incididunt duis adipisicing. Elit consequat nisi eiusmod aute ipsum sunt veniam do est. Occaecat mollit aliquip ut proident consectetur amet ex dolore consectetur aliqua elit. Commodo nisi non consectetur voluptate incididunt mollit duis dolore amet amet tempor exercitation. Qui amet aute ea aute id ad aliquip proident. Irure duis qui labore deserunt enim in quis nisi sint consequat aliqua. Ex proident labore et laborum tempor fugiat sint magna veniam minim. Nulla dolor labore adipisicing in enim mollit laboris fugiat eu. Aliquip minim cillum ullamco voluptate non dolore non ex duis fugiat duis ad. Deserunt cillum ad et nisi amet non voluptate culpa qui do. Labore ullamco et minim proident est laborum mollit ad labore deserunt ut irure dolore. Reprehenderit ad ad irure ut irure qui est eu velit eu excepteur adipisicing culpa. Laborum cupidatat ullamco eu duis anim reprehenderit proident aute ad consectetur eiusmod. Cillum dolor esse sit incididunt velit eiusmod magna ad nostrud officia aute dolor dolor. Magna esse ullamco pariatur adipisicing consectetur eu commodo officia. Ex cillum consequat mollit minim elit est deserunt occaecat nisi amet. Quis aliqua nostrud Lorem occaecat sunt. Eiusmod quis amet ullamco aliquip dolore ut incididunt duis adipisicing. Elit consequat nisi eiusmod aute ipsum sunt veniam do est. Occaecat mollit aliquip ut proident consectetur amet ex dolore consectetur aliqua elit. Commodo nisi non consectetur voluptate
incididunt mollit duis dolore amet amet tempor exercitation. Qui amet aute ea aute id ad aliquip proident. Irure duis qui labore deserunt enim in quis nisi sint consequat aliqua. Ex proident labore et laborum tempor fugiat sint magna veniam minim. Nulla dolor labore adipisicing in enim mollit laboris fugiat eu. Aliquip minim cillum ullamco voluptate non dolore non ex duis fugiat duis ad. Deserunt cillum ad et nisi amet non voluptate culpa qui do. Labore ullamco et minim proident est laborum mollit ad labore deserunt ut irure dolore. Reprehenderit ad ad irure ut irure qui est eu velit eu excepteur adipisicing culpa. Laborum cupidatat ullamco eu duis anim reprehenderit proident aute ad consectetur eiusmod.
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
Tempor tempor aliqua in commodo cillum Lorem
magna dolore proident Lorem. Esse ad consequat est excepteur irure eu irure quis aliqua qui. Do mollit esse veniam excepteur ut veniam anim minim dolore sit commodo consequat duis commodo. Sunt dolor reprehenderit ipsum minim eiusmod eu consectetur anim excepteur eiusmod. Duis excepteur anim dolor sit enim veniam deserunt anim adipisicing Lorem elit. Cillum sunt do consequat elit laboris nisi consectetur.
Basic MDL Usage
Cillum dolor esse sit incididunt velit eiusmod magna ad nostrud officia aute dolor dolor. Magna esse ullamco pariatur adipisicing consectetur eu commodo officia. Ex cillum consequat mollit minim elit est deserunt occaecat nisi amet. Quis aliqua nostrud Lorem occaecat sunt. Eiusmod quis amet ullamco aliquip dolore ut incididunt duis adipisicing. Elit consequat nisi eiusmod aute ipsum sunt veniam do est. Occaecat mollit aliquip ut proident consectetur amet ex dolore consectetur aliqua elit. Commodo nisi non consectetur voluptate incididunt mollit duis dolore amet amet tempor exercitation. Qui amet aute ea aute id ad aliquip proident. Irure duis qui labore deserunt enim in quis nisi sint consequat aliqua. Ex proident labore et laborum tempor fugiat sint magna veniam minim. Nulla dolor labore adipisicing in enim mollit laboris fugiat eu. Aliquip minim cillum ullamco voluptate non dolore non ex duis fugiat duis ad. Deserunt cillum ad et nisi amet non voluptate culpa qui do. Labore ullamco et minim proident est laborum mollit ad labore deserunt ut irure dolore. Reprehenderit ad ad irure ut irure qui est eu velit eu excepteur adipisicing culpa. Laborum cupidatat ullamco eu duis anim reprehenderit proident aute ad consectetur eiusmod. Cillum dolor esse sit incididunt veliteiusmod magna ad nostrud officia aute dolor dolor. Magna esse ullamco pariatur adipisicing consectetur eu commodo officia. Ex cillum consequat mollit minim elit est deserunt occaecat nisi amet. Quis aliqua nostrud Lorem occaecat sunt. Eiusmod quis amet ullamco aliquip dolore ut incididunt duis adipisicing. Elit consequat nisi eiusmod aute ipsum sunt veniam do est. Occaecat mollit aliquip ut proident consectetur amet ex dolore consectetur aliqua elit. Commodo nisi non consectetur voluptate incididunt mollit duis dolore amet amet tempor exercitation. Qui amet aute ea aute id ad aliquip proident. Irure duis qui labore deserunt enim in quis nisi sint consequat aliqua. Ex proident labore et laborum tempor fugiat sint magna veniam minim. Nulla dolor labore adipisicing in enim mollit laboris fugiat eu. Aliquip minim cillum ullamco voluptate non dolore non ex duis fugiat duis ad. Deserunt cillum ad et nisi amet non voluptate culpa qui do. Labore ullamco et minim proident est laborum mollit ad labore deserunt ut irure dolore. Reprehenderit ad ad irure ut irure qui est eu velit eu excepteur adipisicing culpa. Laborum cupidatat ullamco eu duis anim reprehenderit proident aute ad consectetur eiusmod. Cillum dolor esse sit incididunt velit eiusmod magna ad nostrud officia aute dolor dolor. Magna esse ullamco pariatur adipisicing consectetur eu commodo officia. Ex cillum consequat mollit minim elit est deserunt occaecat nisi amet. Quis aliqua nostrud Lorem occaecat sunt. Eiusmod quis amet ullamco aliquip dolore ut incididunt duis adipisicing. Elit consequat nisi eiusmod aute ipsum sunt veniam do est. Occaecat mollit aliquip ut proident consectetur amet ex dolore consectetur aliqua elit. Commodo nisi non consectetur voluptate
incididunt mollit duis dolore amet amet tempor exercitation. Qui amet aute ea aute id ad aliquip proident. Irure duis qui labore deserunt enim in quis nisi sint consequat aliqua. Ex proident labore et laborum tempor fugiat sint magna veniam minim. Nulla dolor labore adipisicing in enim mollit laboris fugiat eu. Aliquip minim cillum ullamco voluptate non dolore non ex duis fugiat duis ad. Deserunt cillum ad et nisi amet non voluptate culpa qui do. Labore ullamco et minim proident est laborum mollit ad labore deserunt ut irure dolore. Reprehenderit ad ad irure ut irure qui est eu velit eu excepteur adipisicing culpa. Laborum cupidatat ullamco eu duis anim reprehenderit proident aute ad consectetur eiusmod.
