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Introduction

Intercellular Communication and DNA Messaging

Intercellular communication between cells in nature allows for coordinated population-level behavior, enabling spatial and temporal organization and complex responses to environmental stimuli. Synthetic biology is continually expanding the range of programmable cellular characteristics and behaviors, and incorporation of intercellular communication into engineered cell populations has extended programmable behavior to a population level.

Engineered AHL-based intercellular communication

Many bacteria naturally secrete acylated homoserine lactone molecules, or AHLs, which can be detected by other members of the bacterial population. Concentration-sensitive detection of AHL brings about significant qualitative changes in cell behavior via transcription regulation, including feedback on AHL production. Such natural quorum sensing systems are associated with coordinated behaviours such as biofilm formation [1] and bioluminescence [2]. These natural bacterial quorum-sensing systems have been successfully modulated to enable programmed intercellular communication in engineered bacterial populations. In this approach, genes associated with AHL production, detection, and response are “re-wired” such that they correspond to different input stimuli and output behaviors. Coordinated population-level behaviors including two-dimensional pattern formation [3], coordinated oscillations in gene expression [4], and even a system exhibiting predator-prey dynamics [5] have been demonstrated using this technique.

Why DNA messaging?

While AHL-based communication is a useful approach for engineering population-level coordination of bacterial cells, the quorum sensing messaging system has some inherent weaknesses that limit the diversity and information content of messages that can be communicated using this method.

Baseballs and Bottles

An analogy is useful in appreciating the expansion of potential for communication afforded by DNA messaging over AHL based messaging. Consider the following absurd but illustrative situation:

The Nuts and Bolts of DNA Messaging

Several key aspects of the M13 filamentous bacteriophage were harnessed by Ortiz and Endy [6] in their original design of the DNA messaging system. Tricking M13 proteins into packaging heterologous DNA: In nature, M13 viral proteins package the M13 genome into viral particles. The M13 DNA is recognized through the M13 packaging sequence. Any DNA containing this packaging sequence will be packaged into viral particles. By removing the M13 packaging sequence from the M13 genome and placing it on a plasmid, we can trick the viral proteins produced from the M13 genome into packaging the plasmid DNA instead of the M13 DNA. A plasmid carrying the packaging sequence is called a phagemid, and a version of the M13 genome that does not efficiently package itself due to a reduced or removed packaging sequence is called a helper plasmid. A phagemid will be packaged into a viral particle in the presence of a helper plasmid. DNA of arbitrary length can be packaged: M13 viral packaging occurs at the cell membrane, where the viral particle forms around the DNA as it is packaged and slowly secreted, forming a long filament. This allows DNA of arbitrary length to be packaged [7]. M13 is not lytic: Since M13 viral particles are secreted through the cell membrane, infected cells are able to continue living and dividing, albeit at ½ to ¾ their normal rate [7]. Because of this, cells sending a DNA message need not commit suicide to transmit their message!

Only F+ cells can be infected: The M13 bacteriophage must attach to the F pilus of an E. coli cell in order to infect it. Therefore, only E. coli cells carrying the F plasmid (F+ cells) are susceptible, while F- cells are not. Sender cells contain a messaging phagemid and a helper plasmid, which allows them to secrete viral particles. Receiver cells must be F+. When the two cell populations are co-cultured, DNA messaging will take place. (Fig 1)

Fig 1 A sender population carrying a messaging phagemid and a helper plasmid can transmit a DNA message to an F+ receiver plasmid in co-culture.

DNA messaging was first established in 2012 when Ortiz and Endy demonstrated transmission and receipt of a DNA message encoding GFP and ampicillin resistance, as well as a separate message encoding T7 RNA polymerase [6]. This proof of principle demonstration indicates the viability of DNA messaging and suggests extension of the method to diversify potential communication programs.

As we can see when the probability of leakiness is on a magnitude of 10^-3, it is well behave as in the secondary modified cells are not as present since there are not a lot of secondary phagemid to infect the cells. However, as shown above, when the magnitude is about 10^-2, it seems as though there are more secondary phagemids that can infect the secondary cells which would cause a large concentration of the secondary modified cell. Thus as we have seen in this analysis that there are some questions that can be solve however many questions can be asked since there may be improvements to the model or the initial parameters themselves.

The Idea:

We are fascinated by the idea of DNA messaging. Since this intercellular communication method [6] is so new, there is room for advancement of the method. We have identified ways to advance the system. We believe that in advanced DNA messaging, a DNA message should be:

1. Controllable: A DNA message should not necessarily be constitutively transmitted, but should rather be controllable by a particular stimulus. 2. *Modifiable: Established methods of DNA recombination should be available for modification of a DNA message by senders or receivers. In particular, recent advances in DNA digital memory and logic [X,X] should be incorporated into DNA messaging. 3. Retransmittable: A receiver cell should be able to retransmit a DNA message following modification.

We developed designs, models, and experiments to approach these goals.

* It should be noted that modification of a DNA message was accomplished by Bonnet et al in a March publication [8]. We were not aware of this until our project was nearly complete. However, our work toward modification of a DNA message was key to our project and did allow us to contribute new BioBricks to the registry.

1. Lewis-Sauer K, Camper A, Ehrlich G, Costerton J, Davies D. Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. Journal of Bacteriology, 2002, 184 (4) pp 1140–54.

2. Nealson K, Platt T, Hastings JW. The cellular control of the synthesis and activity of the bacterial luminescent system. Journal of Bacteriology, 1970, 104 (1) pp 313–22.

3. Basu S, Gerchman Y, Collins CH, Arnold FH, Weiss R. A synthetic multicellular system for programmed pattern formation. Nature, 2005, 434 pp 1130–1134.

4. Danino T, Mondragón-Palomino O, Tsimring L, Hasty J. A synchronized quorum of genetic clocks. Nature, 2010, 463 pp 326–330.

5. Balagaddé FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH, Quake SR, You L. A synthetic Escherichia coli predator–prey ecosystem. Molecular Systems Biology, 2008, 4, pp 1–8.

6. Ortiz ME, Endy D. Engineered cell-cell communication via DNA messaging. Journal of Biological Engineering, 2012, 6:16.

Serine Integrase

The design for accomplishing our goals uses the site-specific recombinase activity of serine integrase enzymes found naturally in various bacteriophages. In nature, serine integrases (Int) allow phages to integrate into the bacterial genome through site-specific recombination between attachment sites on the phage and bacterial genomes (attP and attB), both of which are between 30 and 60 bp depending which phage serine integrase is considered. Recombination between attP and attB leaves behind left and right attachment sites (attL and attR) (Fig 1), which cannot be acted upon by Int alone. Thus, in the presence of Int alone, this recombination is irreversible [12]. In order for the phage genome to excise from the bacterial genome, recombination between attL and attR sites must occur; this reproduces the original phage and bacterial genomes. To this end, phages that exploit serine integrases also have a “recombination directionality factor” (RDF) which reverses the recombination activity of Int; that is, coexpression of Int and RDF allows recombination between attL and attR sites to reproduce attP and attB sites, and inhibits recombination of attP and attB (Fig.2) [12].

Figure 2. Recombination between attP and attB leaves behind attL and attR sites and allows for integration of the phage genome into the bacterial genome in nature. RDF reverses this activity. Adapted from Groth and Calos, 2004 [12].

M13 Bacteriophage

M13 Bacteriophage

The M13 bacteriophage infects E. coli by attaching to the F pilus and injecting single-stranded DNA into the cell. Because the mechanism requires the F pilus, only E. coli cells carrying the F plasmid (F+ cells) can be infected.

M13 bacteriophage is routinely used to isolate single stranded DNA by “tricking” the M13 viral proteins into packaging heterologous DNA that carries the M13 viral packaging site. This site, called the M13 ori, is necessary and sufficient for packaging of circular DNA carrying the site into M13 viral particles [7]. Heterologous DNA packaged this way can still be delivered to an F+ cell and the cell will maintain the circular DNA as a plasmid. A plasmid carrying the M13 ori is termed a “phagemid”. This aspect of M13 bacteriophage is used in DNA messaging.

Importantly, cells infected with M13 bacteriophage do not lyse. Rather, the bacteriophage is secreted through the cell membrane and the cell continues to grow at ½ to ¾ its normal rate [7].

In our attempt to design a system that regulates DNA messaging by controlling M13 particle production, we had the option of manipulating any of the 11 genes in the M13 genome.

M13’s relatively small genome can be classified in three subsets: structural (genes III, and VI - IX), morphogenetic (genes I, IV, and XI), and replicative (genes II, V, and X). The main function behind each of the proteins can be seen in the table below:

Protein:	Function:
pI	Spans the inner membrane of infected bacteria; interacts with viral pIV and host’s thioredoxin; potentially initiates phage assembly [1, 9, 10]
pII	Nicks at specific site in intergenic region of + strand of replicative form (RF) DNA, starting rolling-circle replication; cleaves single-stranded (ss) product of rolling-circle replication; required for the initiation of fl RF DNA synthesis [2, 10]
pIII	Minor coat protein; required for adsorption of phage to sex pili of new hosts [1]
pIV	Resides mainly on outer membrane of infected cell; interacts with pXI and pI within the perisplasm in order to form a gate channel for secretion [1, 9, 10]
pV	Sequesters the viral strands displaced by the rolling circle replication of + strand DNA to prevent their reconversion to double strands [3-7]; needed for the accumulation of exportable ssDNA; controls the rate of synthesis of pII and pX [8]
pVI	Minor coat protein located at proximal end of filament [1]
pVII	Coat protein that interacts with packaging signal in intergenic region [1]
pVIII	Major coat protein; ~2700 copies formed in cylindrical sheath around DNA [1, 11]
pIX	Minor coat protein, located at end of phage particle where assembly begins [1]
pX	Formed from gX, a subset of gII; required for accumulation of ssDNA; powerful repressor of phage-specific DNA synthesis in vivo; limits number of RF molecules [1]
pXI	Aka pI*; forms gate channel in cell membrane along with pI and pIV [1, 9, 10]

The M13 genome takes on two forms: the double stranded replicative form (RF) and the single stranded infective form (IF). When the IF form is initially deposited into the cell, the complementary strand is synthesized by host-encoded enzymes forming the RF form. Protein II nicks the RF form and initiates rolling circle replication, producing additional single stranded copies of the genome that are again converted into RF from by host-encoded machinery. During this process, the M13 genome increases in copy number and viral proteins accumulate in the cell. When protein V reaches a critical concentration in the cell, it binds strongly and cooperatively to single stranded IF DNA, sequestering it to be packaged into viral particles and preventing further accumulation of the RF form [7].

Alternative design comments

The strong affinity of protein V for single-stranded IF DNA is a concern for our DNA message retransmission design, wherein the primary receiver cells have a helper plasmid with gene VIII knockout and the incoming message carries gene VIII under control of a switch. Since gene V is found on the knockout helper plasmid in the primary receiver, it may be present at very high levels in the primary receiver when the DNA message is delivered. The DNA message will be introduced as single stranded DNA, and protein V may bind it, sequester it, and prevent it from replicating. Indeed, it is known that the presence of protein V alone in a cell renders it immune to subsequent infection by M13 bacteriophage [8]. It has also been shown that the presence of gene II in the system reverses this immunity, which demonstrates some regulatory role by gene II.

This suggests that levels of proteins II and V may be relevant to our goal of retransmission of a DNA message. One idea would be to knock out gene V or both genes II and V in the helper and use them as the genes that are complimented. Since proteins II and V are important regulatory proteins, fiddling with them might require extensive remodeling of the M13 regulatory system, which was outside the scope of what we thought was reasonable for our iGEM team. However, it might be wise for those attempting similar goals in the future to consider potential complications arising from proteins II and V.

References

[1] Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbour, NY. 2001

[2] Baas, P. D. (1985). Biochim. Biophys. Acta. 825, 111-139

[3] Mazur, B. J. & Model, P. (1973). J. Mol. Biol. 78, 285-300.

[4] Webster, R. E. & Cashman, J. S. (1973). Virology. 55, 20-38.

[5] Mazur, B. J. & Zinder, N. D. (1975). Virology. 68, 490-502.

[6] Geider, K. & Kornberg, A. (1974). J. Biol. Chem. 249, 3999-4005.

[7] Salstrom, J. S. & Pratt, D. (1971). J. Mol. Biol. 61, 489-501.

[8] Fulford, W. "Bacteriophage F1 DNA Replication Genes *1II. The Roles of Gene V Protein and Gene II Protein in Complementary Strand Synthesis." Journal of Molecular Biology 203.1 (1988): 39-48. Print.

[9] Haigh, Nora G., and Robert E. Webster. "The pI and pXI Assembly Proteins Serve Separate and Essential Roles in Filamentous Phage Assembly." Journal of Molecular Biology 293 (1999): 1017–1027.

[10] Russel, Marjorie. "Interchangeability of Related Proteins and Autonomy of Function: The Morphogenetic Proteins of Filamentous Phage f1 and IKe Cannot Replace One Another." Journal of Molecular Biology 227 (1992): 453-462.

[11] Wickner, William, Gail Mandel, Craig Zwizinski, Marjorie Bates, and Teresa Killick. "Synthesis of phage M13 coat protein and its assembly into membranes in vitro." Proceedings of the National Academy of Sciences 75.4 (1978): 1754-1758.

Design

Controlled Modification of a DNA message

One advantage to DNA messaging is that well-established methods of DNA recombination could potentially be incorporated into intercellular communication programs. We sought to demonstrate this by placing an invertible promoter switch (described below) on a DNA message, which can be flipped using a serine integrase. By placing an invertible promoter switch on a DNA message, we make the message modifiable. The message could be modified in sender cells or receiver cells through production of the serine integrase corresponding to the switch.

Invertible Promoter Switches

Recent work with serine integrases has demonstrated their utility in implementing digital memory and Boolean logic in DNA through inversion of DNA sequences (see page on Serine Integrases). We sought to incorporate an invertible promoter switch into a DNA message, consisting of a promoter whose orientation can be flipped through action of a serine integrase and a corresponding recombination directionality factor (RDF). This would allow the message to take on two different forms depending on the state of the switch. These two forms could correspond to vastly different responses in the receiver population.

Since the switch in the RAD module was demonstrated to be functional, we replicated its architecture exactly in our switches. Intervening spacer sequences between functional elements are also the same in our switches.

Our four switches are portrayed here in PB state (Fig 3).

Fig 3. Schematic diagram of our invertible promoter switches. The orientation of the promoter can be flipped using the integrase corresponding to the att sites in the switch.

Genes placed on either side of the invertible promoter switch will be expressed when the switch is in a state such that the promoter drives their expression. Genes added to the prefix side of the switch will be expressed in PB state, and genes added to the suffix side will be expressed in RL state. Flipping from the PB to RL state and back through action of integrase and RDF is illustrated in figure 4.

Figure 4. The state of an invertible promoter switch can be flipped through the action of integrase and a recombination directionality factor (RDF). Since the promoter is in a different orientation in each state, a different set of genes is expressed depending on the state of the switch.

It is important to note that any genes added to the prefix side of the switch must be inverted relative to the BioBrick convention, i.e. they are transcribed from suffix to prefix, rather than prefix to suffix. These inverted genes are expressed when the switch is in PB state. These switches may be used as a form of passive memory storage. This memory storage is described as passive because it holds its state after the stimulus (Int or Int+RDF) has passed. It does not require active gene expression to hold state, in contrast to switches that function through regulation of transcription (eg a mutual inhibition toggle switch [17]). It should also be noted that in our BioBrick switch constructs there is an NheI restriction site outside of the functional switch portion of the BioBrick close to the prefix. This NheI site is an artifact of a design idea we had early in our project’s design phase and does not have a functional role. It is not expected to affect the functioning of the switch.

Controlled Transmission of a DNA Message

Setting out to design a system that regulates DNA messaging by controlling M13 particle production, we had the option of manipulating any of the 11 genes in the M13 genome (see the M13 Bacteriophage page). In our design, viral particle production is regulated by controlling production of M13 protein VIII. The protein product of M13 gene VIII, protein VIII, is the major coat protein of the M13 phage; wild-type viral particles contain ~2700 copies [18], though this will vary based on the length of the DNA being packaged. The choice to control gene VIII was made based on several factors:

A similar goal was previously accomplished in bacteriophage ϕX174 using the same approach. The gene encoding the ϕX174 major coat protein was removed from the viral genome and placed under an inducible promoter on a plasmid, and induction of this gene was successfully used to control production of ϕX174 viral particles [19].

Gene VIII is an important structural component of the phage particle, but it is not known to have any regulatory function in the M13 genome. Any genes involved in regulation might be more difficult to manipulate while preserving the function of the system.

Gene VIII is the last gene in an operon on the M13 genome. Removing it from the genome is therefore less likely to affect the expression or regulation of other genes.

To control gene VIII expression, we sought to remove gene VIII from the helper plasmid and place it on the messaging phagemid under the control of an inducible promoter. When gene VIII is expressed in the presence of the helper plasmid with a gene VIII knockout, complementation of gVIII occurs and all the viral proteins necessary for phage particle production are present. In this case we expect the messaging phagemid to be sent as a DNA message. When expression of gene VIII is not induced, we expect to see no viral particle production and no DNA messaging.

Controlled Modification and Retransmission of a DNA Message

We sought to combine control over transmission and modification of a DNA message to allow receiver cells to modify a received DNA message and then retransmit it. In the scheme we developed, there are three populations of E. coli that will be co-cultured: the sender population, which transmit a DNA message; the primary receiver population, which receive a message and in the presence of a particular stimulus modify it and retransmit it; and a secondary receiver population, which exhibit a detectable behavior (ie fluorescent protein production) when they receive a modified message (Fig 5).

Figure 5. In our scheme, the sender population sends a DNA message that is received by the primary receiver population. In the presence of a particular stimulus, the primary receiver population modifies the DNA message by flipping an invertible promoter switch and retransmits it. A secondary receiver population will exhibit a detectable behavior (eg fluorescent protein expression) when it receives the modified message.

Reduction in Unwanted Flipping of an Invertible Promoter Switch Using a Double Switch System

A major difficulty in controlling any cellular behavior using gene expression from an inducible promoter is the issue of basal expression; that is, expression of a gene driven by an inducible promoter in the absence of induction. When an invertible promoter switch is controlled through induced expression of integrase, unwanted flipping of the switch due to basal expression of integrase is of particular concern because invertible promoter switches hold their state. To improve control over the flipping of a DNA switch, we devised a double switch system in which a first switch controls the flipping of a second switch. This system, described below, is expected to reduce flipping of a DNA switch due to leaky integrase expression.

References

Videos

Accomplishments

The Waterloo iGEM team accomplished several milestones:

1. The Waterloo iGEM Wet Lab team submitted several Biobricks to the expanding repository of Parts Registry out of which 5 were characterized. Out of the Biobricks submitted, 2 sets of invertible promoter switches, dependent upon viral recombination mechanisms, were submitted. These invertible promoter switches have also been assembled into testable constructs, which utilize different fluorescent proteins for an easy detectable system. These promoter switches can be used to create more complex systems of communication between bacterial cells as opposed to a standard AHL-based system. Each invertible promoter switch has been submitted with its corresponding Integrases and Recombination Directionality factors. Other Biobricks include HPdO, HPdO with deleted gene VIII, and gene VIII. These Biobricks contain M13 viral genes but without the M13 packaging sequence.
2. The mathematical modeling team modeled the dynamics of Bxb1-style and PhiC31-style invertible promoter switch system and controlled modification and retransmission of a DNA message using differential equations, estimating parameter values and analyzing through simulations.
3. The Waterloo iGEM HP team created an educational series called TIL:Synthetic Biology. These TIL episodes teaches interested viewers from the concepts of Synthetic Biology, fundamentals of DNA messaging (Waterloo iGEM’s main project), to perception of Synthetic Biology of the students, academics and philosophy professors.
4. The Waterloo Human Practices team successfully hosted an “Intent to Invent” seminar, where 3 key speakers gathered together to inform students about the applications of synthetic biology in diverse disciplines
5. The Waterloo Human Practices team created a sandbox/launch pad for Biotechnology and Synthetic Biology start-ups known as “Velocity Science”. This marks the beginning of a biotechnology-driven entrepreneurship opportunity targeting primarily the students at the University of Waterloo.

Future Aspirations

Potentially, E.coli strains such as HB101, do not have a prophage at the phi80 locus in their chromosome and could be used in a single copy as opposed to a multi-copy test. Transformation of the pInt80-649 helper plasmid harbouring a temperature sensitive replication of origin (SCI101) and the lambda pir gene into HB101 competent cells can be done. These cells are made competent again for a subsequent co-transformation. Additionally, the switch constructs can be subcloned into an integrative plasmid, pBBIntPhi-J23118, that has a lambda pir origin of replication (R6K). The HB101 competent cells containing pInt80-649, can thus be transformed with sub cloned pBBIntPhi-J23118 and the switch constructs. This will assure the propagation of the integrative plasmid as it has the lambda pir protein being made from the helper plasmid all in one culture. Clones can then be grown in liquid media at 37°C to inactivate the temperature sensitive plasmid. Clones will then be further streaked purified and grown at 43°C over night. This would select for clones that should theoretically only have the integrative plasmid containing the insert of interest (the switches flanked by reporter genes). To confirm proper integration of insert into the bacterial chromosome, colony PCR could be performed on several clones of each switch construct. Ideally, using the primers provided by Zucca et al., 2013 would help anneal upstream of the Phi80 chromosomal attB site and in the R6K origin respectively. The expected amplicon would be 452bp and would indicate the correct integration position, while negative clow would show no amplicon. Additionally, to identify multiple tandem copies of the integrated switches flanked with reporter genes, can be achieved by using another pair of primers also provided by Dr. Zucca. This pair of primers would anneal in opposite directions in the R6K origin of replication and in the upstream region of the cloning sire. A 572 bp amplicon would prove that atleast two tandem copies are present in the genome.

Experiments

Characterization of PhiC31 and Bxb1 invertible promoter switches

Overview

PhiC31 and Bxb1 invertible promoter switches were characterized using test constructs which utilize differential fluorescent protein expression to distinguish between the directionality of the promoter in its PB state and the RL states. These two states are determined by the expression of phage-derived site-specific recombinase, integrase, as well as their corresponding recombination directionality factor or RDF. As outlined in the project description, PhiC31 or Bxb1 integrase alone mediates site-specific recombination between attachment (att) sites (attP and attB sites specifically), flanking each promoter switch in its PB state. Upon recombination, the orientation of the promoter within each switch is reversed and the switch is now in its RL state due to the production of recombinant product sites, attR and attL. Once the switch is in its RL state, expression of PhiC31 and Bxb1 RDFs ,in conjunction with PhiC31 and Bxb1 integrase expression, allows the reversal of the invertible promoter switch back to its PB state. The RDF reverses the directionality of the site-specific recombination mediated by integrase. Designing constructs which allow the expression of specific fluorescent proteins depending on the direction of the promoter within each promoter switch, helped the development of an easy detectable system for characterizing both the PhiC31 and Bxb1 promoter switches as well as their corresponding integrases and RDFs. The characterization of PhiC31 and Bxb1 promoter switches was carried in the presence of both inducible and non-inducible expression of PhiC31 and Bxb1 integrases and RDFs.

Experimental design

BioBricks

1b. Inverted GFP Biobrick:

This is a GFP sequence with the ORF identical to the sequence of (BBa_E0040), but it is oriented opposite to the BioBrick convention. That is, it is read from suffix to prefix, and the coding strand is the opposite strand compared to most BioBricks. This can be expressed from a similarly “inverted” promoter. The Inverted GFP biobrick consists of the inverted GFP with an upstream RBS. The construct is flanked by iGEM Prefix and Suffix. This Biobrick was characterized by placing it upstream of each PhiC31 and Bxb 1 invertible promoter switch (PB state). GFP expression was confirmed using flow cytometry. For information on the characterization of this biobrick, refer to “Characterization of PhiC31 and Bxb 1 invertible switch constructs (PB state)” in the results sections.

Constructs

2a. Multi-copy test constructs:

Multi-copy test constructs were constructed for the following promoter switches in their PB state (promoter switches flanked by attP and attB sites):

1.

1. BBa_K1039001: Bxb1 Invertible Promoter Switch (Promoter J23119) – PB State

2.

2. BBa_K1039008: PhiC31 Invertible Promoter Switch (Promoter J23119) – PB State

3.

3.BBa_K1039009: PhiC31 Invertible Promoter Switch (Promoter J23118) – PB State

4.

The gene for green fluorescent protein (GFP) was placed downstream of each promoter switch in its inverted orientation (BBa_K1039015). This allows for the promoter switch to transcribe GFP in its PB state. The gene for red fluorescent protein (RFP) (BBa_J04450) was placed upstream of each promoter switch and was therefore transcribed only upon a directionally regulated site-specific recombination event on the attP and attB sites flanking the promoter switch. As mentioned before, this event is mediated by PhiC31 and Bxb1 integrases respectively and thereby results in the reversed orientation of the promoter (Fig. 1).

The multi-copy test constructs were assembled via 3A assembly (Parts Registry Assembly Protocol) and sub-cloned into a Parts Registry plasmid, pSB4A5. Each test construct is flanked by the standard iGEM suffix and prefix. The multi-test constructs made include:

1) Bba_K1039025: Test construct for Bxb 1 Invertible Promoter switch (Promoter J23119).

2) Bba_K1039027: Test construct for PhiC31 Invertible Promoter switch (Promoter J23119).

3) Bba_K1039028: Test construct for PhiC31 Invertible Promoter switch (Promoter J23118).

Figure 1. Invertible promoter switch test construct (PB state): The figure above shows the test constructs made for the PhiC31 and Bxb 1 invertible promoter switches in their PB state. The gene for GFP (with its ORF in a reverse orientation) was cloned downstream of each promoter switch. The gene for RFP was cloned upstream of each promoter switch. The invertible promoter switch is flanked by attP and attB sites (as represented by the triangles). The promoter within each promoter switch would drive the expression of GFP in the PB state. Test constructs for all Invertible promoter switches are flanked with iGEM Prefix and Suffix (as indicated by the box labeled “P” and “S”).

Figure 2. Invertible promoter switch test constructs (RL state): The figure above shows the test constructs for the PhiC31 and Bxb 1 invertible promoter switches in their RL state. The gene for GFP (with its ORF in a reverse orientation) was cloned downstream of each promoter switch. The gene for RFP was cloned upstream of each promoter switch. The invertible promoter switch is flanked by attR and attL sites (as represented by the triangles). The promoter within each promoter switch would drive the expression of RFP in the RL state. Test constructs for all Invertible promoter switches are flanked with iGEM Prefix and Suffix (as indicated by the box labeled “P” and “S”).

2b. Multi-copy Non-inducible Integrase-expressing constructs:

Two constructs for the constitutive expression of PhiC31 and Bxb1 integrases respectively, were assembled via Standard Assembly (Parts Registry Assembly Protocol). Genes encoding the PhiC31 and Bxb1 integrases (BBa_K1039012 and BBa_K1039003) were separately cloned downstream of a constitutive promoter and its corresponding ribosome binding site (K608002) in pSB1C3 (Parts Registry Standard vector) (Fig. 2). Each test construct is flanked by the standard iGEM suffix and prefix. The multi-test non-inducible integrase-expressing constructs include the following:

1) Bba_K1039030: PhiC31 integrase-expressing construct with a Promoter and RBS

2) Bba_K1039029: Bxb 1 integrase-expressing construct with a promoter and RBS.

2c. Multi-copy Non-inducible Integrase and Recombination directionality factor- expressing constructs:

Constructs were assembled to allow constitutive expression of both integrase and recombination directionality factor simultaneously from a single promoter. This construct was assembled via Standard Assembly (Parts Registry Assembly Protocol) for both PhiC31 and Bxb1 integrases/RDFs respectively (Fig. 3). Each construct is flanked by the standard iGEM suffix and prefix. The multi-test non-inducible integrase and RDF producing constructs include:

1) Bba_K1039014: PhiC31 integrase and RDF expressing construct from a single promoter. Both genes have an associated ribosome binding site.

2) Bba_K1039007: Bxb 1 integrase and RDF expressing construct from a single promoter. Both genes have an associated ribosome binding site.

Figure 3. Multi-test non-inducible constructs for PhiC31 and Bxb 1 integrase/RDF: (A.) Construct for non-inducible expression of integrase driven from a constitutive promoter and associated RBS for both Phi C31 and Bxb 1 integrase (B.) Construct for non-inducible simultaneous expression of integrase and RDF from a constitutive promoter. Both genes have an associated RBS. All constructs are flanked with iGEM Prefix and Suffix (as indicated by the boxes labeled as “P” and “S”).

2d. Inducible Integrase-Expressing constructs:

The inducible expression of the PhiC31 and Bxb 1 integrase genes was achieved via the use of riboregulators that allow post-transcriptional control of gene expression. Riboregulators include two RNA elements; a cis repressor molecule (also known as the Ribo “lock”) and a trans-activating RNA molecule (also known as the Ribo “Key”). The cis-repressor element is a sequence complementary to the RBS associated with the gene whose expression needs to be silenced (that would be the integrase gene in this case). To control expression of the integrase gene, a Ribo lock was cloned directly upstream of the integrase gene and its associated RBS (5’ UTR). The sequence encoding the Ribo lock, integrase and its associated RBS as well as a lacI cassette (BBa_Q04121) was placed under the control of a strong constitutive promoter (J23119). The trans-activating Ribo key (BBa_I714036) was cloned in the 3’ UTR of the construct and was placed under the control of the PLlac 0-1 promoter (BBa_R0011), which is both LacI-repressible and IPTG-inducible. Simultaneous expression of the LacI cassette and the integrase from a single promoter ensures repression of Ribo key from the LacI promoter and therefore results in a controlled expression of both PhiC31 and Bxb 1 integrase (Isaac et al. 2004).

See uOttawas' Collaboration

1e. Inducible Integrase and Reverse Directionality factor expressing constructs:

Inducible PhiC31 and Bxb 1 Integrase and Reverse Directionality factor expressing constructs were also achieved via the use of riboregulators. As mentioned in section 1d., controlled expression of integrase was achieved via a cis-repressor Ribo Lock and a trans-activating Ribo Key. The gene for RDF was cloned under the control of a LacI promoter (BBa_R0011) with a Ribo lock cloned directly upstream (5’ UTR). For simultaneous expression of both integrase and RDF, the inducible integrase-expressing construct described in section (1d.), was modified to also contain RDF under inducible conditions (Isaac et al. 2004).

See uOttawas' Collaboration

Figure 4. (A.) Inducible PhiC31 and Bxb 1 Integrase-expressing construct: The gene for each integrase is placed under the control of a strong constitutive promoter (J23119) with a Ribo “lock” directly upstream. The trans-activating Ribo “key” is placed downstream under the control of a PLlac 0-1 promoter. The lacI cassette is also placed under the control of promoter J23119. (B.) Inducible PhiC31 and Bxb 1 integrase and recombination directionality factor-expressing construct: The genes for the integrase and the RDF were placed directly downstream from their corresponding ribo “lock” with the integrase under the control of a strong constitutive promoter and the RDF under the control of a PLlac 0-1 promoter. The lacI cassette (BBa_Q04121) was placed under the control of a strong constitutive promoter (J23119).

Results and Conclusions

Characterization of PhiC31 and Bxb 1 invertible promoter switches (PB state)

PhiC31 and Bxb1 invertible promoter switches in their PB state drove the expression of GFP. GFP expressing colonies for each invertible promoter test constructs growing on the LB agar plate was detected using a UV filter. Upon detection, one GFP-expressing colony for each test construct was selected and inoculated in 5mL of LB (with appropriate antibiotics) and grown overnight at 37°C. Flow cytometry was then used to confirm GFP expression from all cultures. The results are shown in the figure below. Since a modified version of GFP (ORF was modified to be in the opposite orientation with respect to convention) was used for these constructs, flow cytometry on bacterial cells producing GFP (BBa_I20260) was used as one of the controls. As indicated by the results, GFP expression from both cultures result in a shift in fluorescent populations when compared to control cells expressing no fluorescent protein. These results confirm the proper functioning of our modified GFP gene and furthermore, characterize the promoter switches in their PB state. Once the Invertible Promoter switches were co-transformed with its corresponding integrase producing construct, the resulting colonies were analyzed for RFP expression. This experiment could only be carried out for Bxb 1 invertible promoter switch (Promoter J23119- PB state) due to time constraints. Figure 8 below shows RFP expressing bacterial colonies growing on LB agar with appropriate antibiotics after the co-transformation. As previously mentioned, RFP expression is driven by the Bxb 1 invertible promoter switch (promoter J23119) due to the recombination event mediated by Bxb 1 integrase. RFP expressing cells confirm the proper functioning of the flipped promoter switch (RL state) and further characterizes the Bxb 1 integrase.

Figure 5. Testing PhiC31 and Bxb1 Invertible promoter switches (PB state): To test the invertible promoter switches in their PB state (flanked by attP and attB), they wre independently co-transformed with their corresponding non-inducible integrase-expresin constructs and consequently plated on LB agar with appropriate antibiotics to select for both plasmids. The resulting bacterial colonies were then analyzed for RFP expression.

Figure 6. Characterization of PhiC31 and Bxb 1 Invertible Promoter switch (RL state): To characterize the Bxb 1 invertible promoter switch in its RL state (flipped orientation of the promoter), the Bxb 1 invertible promoter switch (with flipped switch) was isolated from the bacterial colony, which also included an integrase-expressing plasmid. Upon isolation of the Bxb 1 invertible promoter switch construct, it was co-transformed with the constitutive RDF and integrase producing plasmid and grown on an LB agar plate with appropriate antibiotics. The plate was the screened for GFP expression colonies

Figure 7: Flow cytometry for confirming expression of inverted GFP (A) negative control: bacterial cells expressing no fluorescent protein (B) GFP (BBa_I20260) expressing bacterial cells (C) Bacterial cells expressing inverted GFP from the invertible promoter switch test construct. GFP expression from cultures (B) and (C) result in a shift in fluorescent populations when compared to control cells expressing no fluorescent protein (A). These results confirm the proper functioning of our modified GFP gene and furthermore, characterize the promoter switches in their PB state

Figure 8: Characterization of PhiC31 and Bxb 1 invertible promoter switches (PB state): Each of the invertible promoter switch constructs was grown in LB media with appropriate antibiotics (tube 3). The cultures were grown for 16 hours at 37 degrees and centrifuged at 13 000 rpm for 1 minute. The resulting pellet was checked under an appropriate UV filter. Tube 3 can be seen to be expressing adequate amounts of GFP. Tube 1 is centrifuged culture with bacterial cells expressing no fluorescent proteins and tube 2 is centrifuged culture with bacterial cells expressing RFP. Tubes 1 and 2 seem black under the filter.

Figure 9: Characterization of Bxb 1 invertible promoter switch (Promoter J23119): Site-specific recombination event mediated by Bxb 1 integrase produces recombined products: attR and attL. This results in the orientation of the promoter to be flipped. The promoter switch drives the expression of RFP in the RL state. The figure shows Bxb 1 invertible promoter switch test construct co-transformed with a constitutive integrase-expressing construct. When grown on an LB agar plate for 16 hours at 37 degrees with appropriate antibiotics, all bacterial colonies were expressing RFP.

Gene VIII Complementation Test

To accomplish our goal of controlling DNA messaging, we sought to control M13 viral particle production by removing M13 gene VIII from the helper plasmid and placing it under an inducible promoter on a separate construct. M13 protein VIII, the product of gene VIII, is the major coat protein of the M13 bacteriophage, so viral particle production and DNA messaging should be possible only when gene VIII is expressed and protein VIII is present (see the Design page). As a step toward this goal, we first sought to demonstrate that viral particles could be produced when a helper plasmid missing gene VIII is complemented by a copy of gene VIII on a separate construct (See Figure xx)

Figure --: The messaging phagemid (A) contains the M13 packaging sequence, an RFP indicator, and M13 gVIII which will complement the helper plasmid (B), which contains the remaining M13 genes, in order to produce viral particles.

After making our constructs, we set out to determine if our helper plasmid with gene VIII knockout (BBa_K1039017) could be complemented with a messaging phagemid consisting of M13 origin (BBa_K314110) - RFP expression cassette (BBa_J04450) - Constitutive Promoter and Ribosome Binding site + M13 gene VIII (BBa_K1039020) in pSB1C3. We expected gene VIII to be expressed from the messaging phagemid and to complement the helper plasmid with gene VIII knockout. With a complete set of functional proteins, the virus should be able to assemble itself and package the messaging phagemid through recognition of the M13 ori to successfully deliver the message to the receiver population. Chloramphenicol resistance (from the pSB1C3 backbone) and RFP expression should be detectable in receiver cells that have received the DNA message.

To test the complementation of gene VIII, we used E. coli βDH10B containing messaging phagemid and helper plasmid as a sender population, and we used E. coli JM109 containing a constitutively expressed GFP expression cassette (BBa_I20260) in pSB3K3 (kanamycin-resistant) backbone as a receiver population. The senders, E. coli βDH10B, require diaminopimelic acid (DAP) as supplement for growth in LB media. The receivers, E. coli JM109 are F+ and do not require this supplement. (Recall that receivers must be F+ for delivery of a DNA message by an M13 particle.) This allows counter-selection against sender cells by growing on LB media with no DAP supplementation. Receiver cells that have received the message can be selected using chloramphenicol and should also appear red.

Figure --: Schematic representation of M13 complementation test.

Co-culture of sender and receiver populations

Message transmission was tested by co-culturing sender and receiver populations before selecting for successful transmission. During the co-culture, M13 particles containing the DNA message should be secreted by sender cells and should deliver the message to receiver cells.

Exposure of receiver population to filtrate from sender culture

Since the message is transmitted through viral particles, sender cells should not need to be in contact with receivers for message transmission; presence of message-carrying viral particles should be enough. We therefore also tested message transmission by exposing receiver cells to culture filtrate from sender cells, which should contain viral particles secreted by the senders that can deliver messages to receivers.

Results for M13 complementation co-culture experiment in liquid LB Cm Km

For the co-culture complementation tests, we expected growth only in cultures with sender cells that have a complete set of M13 virus structural genes (HPdO helper plasmid with gVIII in the system) and the M13 ori. However, our results showed significant growth in co-cultures containing JM109 (F+) receiver cells and βDH10B (F-) sender cells that did not contain the helper plasmid. The co-cultures that had DH5α as the receiver cells but the βDH10B (F-) sender cells did not show growth. One hypothesis for this phenomenon is the possibility of the receiver cells gaining the messaging phagemid through conjugation rather than through viral infection. On the other hand, co-cultures that contained the complete system necessary to transmit a messaging phagemid grew much slower than the sender cells without helper plasmid mentioned previously. This could be due to the fact that infection by M13 viral particles often slows bacterial growth. There are many variables that must be taken into account when analyzing such results so we propose to repeat the experiment conducted by Ortiz and Endy (2012) who were able to complement HPdO helper plasmid (BBa_K1039016) with a phagemid containing the M13 packaging sequence to transmit the phagemid from a F+ sender population to a F+ receiver population6.

The filtrate experiment was the second experiment proposed by our group and this involves filtering out the sender cells and using their supernatant to infect the receiver cells. Conjugation requires cell-to-cell contact so the filtrate experiment limits the chance of conjugation since none of the sender cells will be present. The filtrate experiment was performed but no colonies were observed on any of the plates; therefore, the experiment must be replicated and optimized for the next trial.

In addition to the experiments proposed above, we devised a plaque assay that allows us to visualize M13 infection of the receiver cells. Although M13 virus does not undergo the lytic cycle, its infection of cell populations slows their growth and thus an area of diminished growth is produced around colonies, creating turbid plaques7. In order to propagate the message received by JM109 cells, we propose to transform helper plasmid with and without gene VIII into these cells. Upon transformation with a messaging phagemid that would complete the system, JM109 would be able to infect neighbouring cells once plated on LB Agar Cm 20 Km 25. This experiment has not been attempted as of yet but it has the potential to provide us with more insight into our proposed system.

Construct name (DH10B)	Biobricks	Optical Density 600nm (OD600)
		Co-culture with JM109 (F+) containing) BBa_I20260	Co-culture with DH5α (F-) containing BBa_I20260
HPdO and pSB1C3-M13 ori- RFP cassette	BBa_K1039016 and BBa_K314110- BBa_J04450	0.039	0.023
HPdO and pSB1C3-RFP cassette	(BBa_K1039016) BBa_J04450	0	0
HPdO Δ gVIII and pSB1C3- M13 ori - RFP- Constitutive promoter + RBS -gVIII	BBa_K1039017 BBa_K314110 BBa_J04450 BBa_K1039020	0.054	0.019
HPdO Δ gVIII and pSB1C3- RFP- Constitutive promoter + RBS - gVIII	BBa_K1039017 BBa_J04450 BBa_K1039020	0.118	0
HPdO Δ gVIII and pSB1C3 - M13 - RFP cassette	BBa_K1039017 BBa_K314110 BBa_J04450	0	0
HPdO	BBa_K1039016	0	0
HPdO Δ gVIII	BBa_K1039017	0	0
pSB1C3- M13 ori - RFP- Constitutive promoter + RBS - gVIII	BBa_K314110 BBa_J04450 BBa_K1039020	0.299	0.014
pSB1C3- M13 - RFP cassette	BBa_K314110 BBa_J04450	0.370	0.011
pSB1C3- RFP- Constitutive promoter + RBS - gVIII	BBa_J04450 BBa_K1039020	0.378	0.022
pSB1C3- RFP cassette	BBa_J04450	0.423	0.038
Blank	No biobricks	0	0

Controls

As a positive control, we used senders carrying a complete helper plasmid (gene VIII present) and a messaging phagemid carrying the M13 origin of replication and an RFP expression cassette (BBa_K314110- BBa_J04450). With the helper plasmid intact and all phage proteins expressed, we expect the phage machinery to recognize the M13 origin and package the messaging phagemid to be sent to the receiver population.

As a negative control to demonstrate the necessity of the M13 origin, we performed an experiment identical to the positive control using a messaging phagemid that is missing the M13 origin. Without the M13 origin, we expect no transmission of the messaging phagemid. We also performed a negative control for the gene VIII complementation experiment wherein the messaging phagemid was missing the M13 ori. As a negative control to demonstrate the necessity of gene VIII for viral packaging, we performed an experiment identical to the gene VIII complementation experiment, but with the messaging phagemid lacking gene VIII (ie no gene VIII was present in the system). As a negative control to demonstrate the necessity of having both a helper plasmid and a messaging phagemid for successful transmission, we also included experiments wherein senders contained only a messaging phagemid or only a helper plasmid. In these cases we expected no message transmission. As a negative control to demonstrate the necessity for F+ receivers, we repeated all experiments using DH5α (F- strain) as the receiver population. We expected no successful messaging with F- receivers, because the F pilus, which is only present in F+ strains, is required for the mechanism of message delivery using M13 particles.

In addition, we plated senders and receivers individually on our selection plates to check for contamination. These controls were used in both the co-culture and filtrate experiments.

Experimental Design

For the purposes of the complementation tests, the following constructs were sequentially transformed into βDH10B:

1. HPdO (BBa_K1039016) and pSB1C3-M13 ori- RFP cassette (BBa_K314110- BBa_J04450) 2. HPdO (BBa_K1039016) and pSB1C3-RFP cassette (BBa_J04450) 3. HPdO Δ gVIII (BBa_K1039017) and pSB1C3- M13 ori - RFP- Constitutive promoter + RBS - gVIII (BBa_K314110- BBa_J04450- BBa_K1039020) 4. HPdO Δ gVIII (BBa_K1039017) and pSB1C3- RFP- Constitutive promoter + RBS - gVIII (BBa_J04450- BBa_K1039020) 5. HPdO Δ gVIII (BBa_K1039017) and pSB1C3 - M13 - RFP cassette (BBa_K314110- BBa_J04450) 6. HPdO (BBa_K1039016) 7. HPdO Δ gVIII (BBa_K1039017) 8. pSB1C3- M13 ori - RFP- Constitutive promoter + RBS - gVIII (BBa_K314110- BBa_J04450- BBa_K1039020) 9. pSB1C3- M13 - RFP cassette (BBa_K314110- BBa_J04450) 10. pSB1C3- RFP- Constitutive promoter + RBS - gVIII (BBa_J04450- BBa_K1039020) 11. pSB1C3- RFP cassette (BBa_J04450)

The following construct was transformed into JM109 and DH5α:

A. pSB3K3- GFP cassette (BBa_I20260) in JM109 strain B. pSB3K3- GFP cassette (BBa_I20260) in DH5α strain

Please refer to Table 1.1 under protocols for antibiotic concentrations.

Please refer to Table 1.1 under protocols for antibiotic concentrations.

• Grow cultures 1-11 and A-B individually in LB with appropriate antibiotic supplements until OD600 ≈ 0.7 (± 0.1) • Centrifuge cultures and wash pellet 3 times with LB • Resuspend 1-11 in appropriately supplemented media but without Cm since the receiver population is not Cm resistant • Resuspend A-B in LB DAP since some of the senders are not Km resistant • Incubate at room temperature with shaking for 6-10 h • Co-culture senders and receivers in LB DAP (Km only for senders 1-7) broth in a 2:1 ratio of senders to receivers to ensure that a sufficient amount of message is present • Incubate at room temperature with shaking for 6-10 h • Centrifuge and wash pellet 3 times with LB to remove DAP from the media • Resuspend in LB Km Cm • Subculture with a dilution factor of 1/100 into LB Km Cm so only receivers carrying the messaging phagemid and the GFP plasmid are able to grow • Incubate over night at 37 °C with shaking • Dilute culture 10-6 times • Plate 100 μL of diluted culture onto LB Cm 20 Km 25 agar

Filtrate experiment

• Grow cultures 1-11 and A-B individually in LB with appropriate antibiotic supplements until OD600 ≈ 0.7 (± 0.1) • Centrifuge cultures and wash pellet 3 times with LB • Resuspend 1-11 in appropriately supplemented media but without Cm since the receiver population is not Cm resistant • Resuspend A-B in LB DAP since some of the senders are not Km resistant • Incubate at room temperature with shaking for 6-10 h • Collect filtrate of senders by centrifuging cultures at room temperature • Filter sterilize through 0.45 μm filter to remove remaining bacterial cells in the filtrate • Infect the receiver population by mixing it with the sender filtrate • Incubate at room temperature for 6-10 h • Plate all cells on LB Cm 20 Km 25 agar

Table here Table here Table here Table here Table here

Genotype of Strains

DH5α F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–

JM109 endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+ Δ(lac-proAB) e14- [F' traD36 proAB+ lacIq lacZΔM15] hsdR17(rK-mK+)

βDH10B F- endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74 Φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) λ-

Plasmids Used

pSB1C3

pSB3K3

HPdO

Plasmids used for Construction

pSB1A3

pSB4A5

BioBricks

Bba_K1039000	Φ attP site
Bba_K1039001	BXB1 pb switch J23119 promoter
Bba_K1039002	BXB1 pb switch J23118 promoter
Bba_K1039003	BXB1 integrase
Bba_K1039004	BXB1 lr switch J23119 promoter
Bba_K1039005	BXB1 lr switch J23118 promoter
Bba_K1039006	BXB1 RDF
Bba_K1039007	BXB1 integrase and RDF
Bba_K1039008	ΦC31 pb switch J23119 promoter
Bba_K1039009	ΦC31 pb switch J23118 promoter
Bba_K1039010	ΦC31 lr switch J23119 promoter
Bba_K1039011	ΦC31 lr switch J23118 promoter
Bba_K1039012	ΦC31 integrase
Bba_K1039013	ΦC31 RDF
Bba_K1039014	ΦC31 integrase and RDF
Bba_K1039015	iGFP
Bba_K1039016	Hpdo backbone
Bba_K1039017	Hpdo with no gene 8
Bba_K1039018	gene 8 of M13 virus
Bba_K1039019	RBS+gene 8 of M13 virus
Bba_K1039020	J23104+RBS+gene 8 of M13 virus
Bba_K1039021	loc+BXB1int+key+lock+BXB1 RDF
Bba_K1039022	loc+PhiC31int+key+lock+phiC31 RDF
Bba_K1039023	loc+BXB1int+key
Bba_K1039024	loc+BXB1int+key
Bba_K1039025	BS1TC
Bba_K1039026	BS2TC
Bba_K1039027	ΦS1TC
Bba_K1039028	ΦS2TC
Bba_K1039029	BXB1 integrase with the Promoter and RBS
Bba_K1039030	ΦC31 integrase with the Promoter and RBS
Bba_K1039031	BXB1 att P
Bba_K1039032	BXB1 att B
Bba_K1039033	BXB1 att R
Bba_K1039034	BXB1 att L
Bba_K1039035	ΦC31 att B
Bba_K1039036	ΦC31 att R
Bba_K1039037	ΦC31 att L

Ottawa's Collaboration

This year Waterloo iGEM collaborated with Ottawa iGEM. Ottawa team required help with Mathematical Modelling. In exchange Ottawa team was able to help us build the following constructs:

Promoter-LacI cassette-lock-Bxb1 integrase-Transcriptional Terminator-Promoter- Key-Transcriptional Terminator-Promoter- Lock- Bxb1 RDF

Promoter-LacI cassette-lock-PhiC31 integrase-Transcriptional Terminator-Promoter- Key-Transcriptional Terminator-Promoter- Lock- PhiC31 RDF

Promoter-LacI cassette-lock-Bxb1 integrase-Transcriptional Terminator-Promoter- Key-Transcriptional Terminator

Promoter-LacI cassette-lock-PhiC31 integrase-Transcriptional Terminator-Promoter- Key-Transcriptional Terminator

Notebook

Population & Infection Modelling

Modelling the Dynamics of Bxb1-style and PhiC31-style Invertible Promoter Switch Systems

Invertible Promoter Switch “Styles”

We sought to investigate the qualitative differences between the dynamics of an invertible promoter switch whose integrase and RDF interactions are similar to the Bxb1 system and one whose integrase and RDF interactions are similar to the PhiC31 system. Notably, the Bxb1 RDF is thought to interact with Bxb1 Int only when Int is bound to DNA at the Bxb1 att sites [1] while PhiC31 RDF is known to complex with Int in the cytosol independently of DNA binding [2]. We refer to a switch system where RDF binds Int only when Int is already complexed with an att site as a “Bxb1-style” system, and we refer to a switch system where RDF can complex with Int independently of DNA as a “PhiC31-style” system.

We extended this comparison of Bxb1-style and PhiC31-style switches to a two-switch system where the state of the second switch is controlled by the state of the first switch (Fig X), as proposed as an alternative design for controlled modification and retransmission of a DNA message (see the Improving Robustness of an Invertible Promoter Switch Using a Double Switch System section on the Project Page). Herein we refer to the first switch as Switch 1 and the second switch as Switch 2. The integrase and RDF acting on Switch 1 are referred to as Int 1 and RDF 1, and those acting on Switch 2 are referred to as Int 2 and RDF 2.

It is important to note that our model required a small presence of RDF 1 in order to prevent complete flipping of all copies of Switch 1 due to basal expression by Int 1. The double switch system is outlined as follows:

• Input to the system is induction of Int 1 from an inducible promoter.

• Constitutive low-level expression of RDF 1 is required to prevent complete flipping of Switch 1 due to basal Int 1 expression. This may be an artifact of our model.

• Switch 1 in PB state transcribes RDF 2

• Switch 1 in RL state transcribes Int 2

• Switch 1 is present in multiple copies

• For Int 2 to overcome the presence of RDF 2, most copies of Switch 1 must be flipped.

• Basal expression of Int 1 may be able to flip a fraction of the copies of Switch 1, but not all, in which case Switch 2 does not flip.

• Induced expression of Int 1 should flip all copies of Switch 1, in which case Switch 2 flips.

Our analysis considers all combinations of Bxb1-style and PhiC31-style switches in such two-switch systems: (Switch 1, Switch 2) = (Bxb1-style, Bxb1-style) or (Bxb1-style, PhiC31-style) or (PhiC31-style, Bxb1-style) or (PhiC31-style, PhiC31-style). In all cases, the two switches are assumed to operate using orthogonal integrase/RDF systems. In the case where the style of the two switches is the same (eg both switches Bxb1-style), it is assumed that we are working with two orthogonal integrase/RDF systems that have the same integrase/RDF interaction pattern.

Questions We would Like to Shed Light On

Our goal in modeling is model-based design. We would like to use our model to guide our design choices to direct lab efforts. We sought to use a model of switch dynamics to shed light on the following questions:

• Which switch style, Bxb1 or PhiC31, is more robust in terms of flipping when we want it to flip and holding state when we want it to hold state?

• What is the increase in robustness obtained by using a two-switch system vs a one-switch system?

• What is an acceptable level of uninduced basal Int 1 expression?

(To cut to the chase, skip to the conclusions section.)

Model Construction

State variables (all time-dependent):

I1 = Integrase acting on Switch 1

R1 = RDF acting with I1 on Switch 1

I2 = Integrase acting on Switch 2

R2 = RDF acting with I2 on Switch 2

mx = mRNA corresponding to protein X.

S1PB = Switch 1 in “PB” state, where we have attP and attB sites flanking the promoter

S1RL = Switch 1 in “RL” state, where we have attR and attL sites flanking the promoter

S2PB = Switch 2 in “PB” state

S2RL = Switch 2 in “RL” state

I2 = integrase dimer

I2SPB = integrase dimer bound to one att site in a PB state switch

I2RXSPB = integrase dimer bound to one att site in a PB state switch, with X RDF proteins also bound (X = 1 or 2)

I4SPB = one integrase dimer bound to each att site in the PB state switch

I4RXSPB = one integrase dimer bound to each att site in the PB state switch, with X RDF proteins also bound (X = 1–4)

In the PhiC31 style switch but NOT the Bxb1 style switch, the following complexes are also included:

I2Rx = X RDF proteins bound to an integrase dimer, X = 1 or 2

Parameters:

αPind = inducible transcription rate from inducible promoter producing Integrase 1

λPind = basal “leaky” transcription rate from inducible promoter producing Integrase 1

αR1 = transcription rate of RDF 1

αS1 = transcription rate from promoter in Switch 1

αS2 = transcription rate from promoter in Switch 2

δx = degradation rate of mRNA corresponding to protein X

βx = translation rate of protein X from mRNA

ρx = degradation rate of protein X

i = concentration of inducer molecule

Ki = half-saturation constant for inducer

k1 = forward rate constant for association of a protein to a complex

k-1 = reverse rate constant for dissociation of a protein to a complex

krec = rate of switch flipping when bound to appropriate complex

Note that rate constants k1 and k-1 are assumed to be the same for formation of all complexes, since rate constants for the complexes are not known. krec is assumed to apply to flipping of the switch from PB to LR state and also from LR to PB state, when DNA is bound in the appropriate complexes.

Differential Equations:

The differential equation for the abundance of an mRNA species is determined by transcription and degradation rates of mRNA. For the inducible promoter producing Integrase 1 that can flip Switch 1 and the constitutive promoter producing a very low level of RDF 1, promoter copy number is assumed constant and the transcription rates α do not depend on DNA concentration. For these mRNAs we have:

For any mRNA species that is transcribed from a switch, the total amount of switch in the correct position affects the rate of transcription. For example, the differential equation for abundance of an mRNA produced from Switch 1 in PB state would look like:

where [S1PBTot] is the total concentration of S1 in PB state, which is the sum of the concentrations of all complexes involving S1PB.

The differential equation for the abundance of a complex X of I, R, and S takes into account the following factors:

• The rate of formation of X from association of any complexes A and B that can form X

• The rate of depletion of X from dissociation back into A and B

• The rate of formation of X from dissociation of any complex Z that dissociates into two species X and Y

• The rate of depletion of X from association of X with a species Y to form a complex Z

• If X is a monomer of I or R, then X is also assumed to be produced through translation

• If X is a complex that can be produced from a complex C through flipping of a switch, then the rate of switch flipping is included

• If X is a complex that can transition into a complex W through flipping of a switch, it can be depleted this way

• Proteins degrade at a rate proportional to the amount of protein.

• DNA is assumed to be conserved, without production or degradation. To allow protein complexes to degrade without losing DNA, degradation of any complex involving DNA is assumed to apply only to the protein and the DNA is left behind.

Thus the general differential equation for abundance of a complex X is:

where n different protein pairs Ai and Bi can complex to form X through the interaction

X can complex with m different proteins Yj to form m complexes Zj through the interaction

and the complex degrades at a rate ρx.

If X is a monomer of I or R, then we also add the production term

where mx is the mRNA corresponding to protein X.

If X is a complex that can be formed from complex C through flipping of a switch, we also add the production term

and X is a complex that transitions to complex W through flipping of a switch, we also add the depletion term

Note that the only reactions involving switch flipping are:

If X is naked DNA, eg S1PB, then DNA that is “lost” in the model through degradation of n complexes involving DNA is assumed to become naked DNA and we add the term,

where Pi are all the complexes involving the DNA X, each degrading at a rate ρx.

Example:

For abundance of integrase dimer, which for a PhiC31 style integrase takes part in the interactions

the differential equation is:

Analysis

With identical parameter values (Parameter set 1, see bottom), we simulated a two-switch system for each potential (Switch 1, Switch 2) combination, with Int 1 induced (Table X) and uninduced with basal Int 1expression level λPind equal to 1/100, 1/75, 1/50, and 1/25 the induced Int 1 expression level (Table X).

Conclusions

Estimation of Parameter Values

αPind = inducible transcription rate from inducible promoter producing Integrase 1

λPind = basal “leaky” transcription rate from inducible promoter producing Integrase 1

αR1 = transcription rate of RDF 1

αS1 = transcription rate from promoter in Switch 1

αS2 = transcription rate from promoter in Switch 2

δx = degradation rate of mRNA corresponding to protein X

βx = translation rate of protein X from mRNA

ρx = degradation rate of protein X

i = concentration of inducer molecule

Ki = half-saturation constant for inducer

k1 = forward rate constant for association of a protein to a complex

k-1 = reverse rate constant for dissociation of a protein to a complex

krec = rate of switch flipping when bound to appropriate complex

References

1. Ghosh P, Wasil LR, Hatfull GF (2006) Control of phage Bxb1 excision by a novel recom- bination directionality factor. PLoS Biol 4:e186.

2. Khaleel, T., Younger, E., McEwan, A. R., Varghese, A. S., & Smith, M. C. M. (2011). A phage protein that binds φC31 integrase to switch its directionality. Molecular microbiology, 80(6), 1450–63. doi:10.1111/j.1365-2958.2011.07696.x

Population & Infection Modelling

Modeling Controlled Modification and Retransmission of a DNA Message

We sought to combine controlled modification and controlled transmission of a DNA message to design a system wherein receiver cells are able to modify a DNA message by flipping an invertible promoter switch and then retransmit the modified message (see the section on Controlled Modification and Retransmission of a DNA Message on the Design Page).

The system is summarized as follows:

• Three populations are present in co-culture: sender cells, primary receiver cells, and secondary receiver cells.

• Senders transmit a DNA message with an invertible promoter switch in PB state. M13 gene VIII along with a T7 RNA polymerase gene are positioned on the message phagemid such that they are expressed when the switch is in LR state.

• Primary receiver cells are F+ and contain a helper plasmid with a gene VIII knockout. Viral particles cannot be produced using protein products from this helper plasmid alone. Primary receivers also contain an inducible integrase gene corresponding to the switch on the DNA message.

• When the switch on the message is flipped in primary receiver cells, gene VIII is expressed and the helper plasmid with gene VIII knockout is complemented. With a full suite of M13 genes, the modified version of the messaging phagemid (Mmod) can be packaged into viral particles and retransmitted.

• Secondary receiver cells are F+ and contain a gene for a fluorescent protein driven by a T7 promoter. When a secondary receiver cell receives a modified messaging phagemid (switch flipped), the T7 RNA polymerase expressed from the message causes detectable fluorescent protein production.

The goal for this system is for secondary receiver cells to receive the modified message phagemid when modification and retransmission by primary receivers is induced. The modified message should not be transmitted in absence of induction.

This system is prone to several undesired outcomes cause by various modes of failure, outlined below:

• Since primary receivers must be infected by Mori before they can produce Mmod, there is a time lag between accumulation of Mori and Mmod. A time lag is also expected in the “turnaround” between reception of a DNA message and accumulation of copy number and protein concentration that can fuel packaging and transmission of the modified message. If the majority of secondary receivers become infected with Mori during this turnaround, Mmod will not have a significant presence in the secondary receiver population.

• We have picked apart the M13 genome in order to allow control over production of viral particles. As a result, it is likely that production of viral particles by primary receivers may be less efficient than particle production in senders. If the rate of particle production is drastically reduced in primary receivers as compared to senders, Mori may accumulate much faster than Mmod throughout the entire experiment

2. Since the integrase that flips the switch in primary receivers is controlled by an inducible promoter, basal (uninduced) expression of integrase may cause unwanted flipping of the switch, which would result in production of Mmod in the absence of induction. Since a switch holds state after flipping, such an error would be irreversible. This problem is exacerbated by the fact that the resulting unwanted message transmission would spread to other cells. Such amplification of error could make the system effectively constitutive, even though an inducible promoter is employed.

Questions We would Like to Shed Light On

Our goal in modeling is model-based design. We would like to use our model to guide our design choices to direct lab efforts. We sought to use a model of the spread of Mori and Mmod through the co-cultured populations to shed light on the following questions:

• What is the minimum efficiency of viral particle production in primary receivers as compared to senders that preserves functionality of the system?

• What ratio of initial populations (senders : primary receivers : secondary receivers) is optimal to accomplish successful delivery of Mmod to the secondary receiver population?

• What is the effect of basal uninduced expression of integrase in primary receivers? To what extent does such error become amplified?

Model Construction

We used the following set of state variables and parameters to model the spread of Mori and Mmod through the co-cultured populations:

State variables (all time-dependent):

Ps = population of sender cells

P1 uninfected = population of primary receiver cells that have not received a DNA message

P2 uninfected = population of secondary receiver cells that have not received a DNA message

P1 ori = population of primary receiver cells carrying the original DNA message

P2 ori = population of secondary receiver cells carrying the modified DNA message

P1 mod (t,a) = population of primary receiver cells of age a at time t. Age a corresponds to how long the cell has been a P1 mod cell. This is important to know because of the turnaround time between reception of a DNA message and accumulation of phagemid and protein to fuel viral particle production.

P2 mod = population of secondary receiver cells carrying the modified DNA message

Mori = concentration of M13 viral particles carrying the original unmodified DNA message

Mmod = concentration of M13 viral particles carrying the modified DNA message

Parameters:

c = carrying capacity of the liquid growth medium

g = maximum growth rate of cells that are not producing viral particles in absence of pressure due to carrying capacity

r = maximum growth rate of cells that are producing viral particles as a fraction of g

k = adsorption rate of viral particles to cells

i = induction constant. i = 1 when message modification and retransmission is induced, and i = 0 when it is not induced

S(i) = rate of switch flipping per primary receiver carrying Mori

p = rate of viral particle production through intact M13 cistrons on the helper plasmid

b = rate of viral particle production through complemented helper plasmid with gene VIII knockout, expressed as a fraction of p

j1 = age at which viral particle production begins in P1 mod cells. Production starts at zero at age j1 and increases.

j2 = age at which viral particle production reaches its maximum and continues to maintain a steady rate

We built a differential equations (DEs) model of the system using these state variables and parameters, which is described below.

When modeling the growth of each population over the course of the experiment, the carrying capacity of the media had to be considered. To simplify our equations, we define:

The DE for senders is a simple logistic growth equation:

where the growth rate G is modified by r since senders produce viral particles and therefore grow more slowly. The DEs for P1 uninfected and P2 uninfected incorporate loss due to receipt of DNA messages, and the DE for P2 ori incorporates production due to receipt of DNA message:

Since there is a turnaround time associated with receipt, modification, and retransmission of a DNA message in primary receivers, the “age” of P1 mod cells – the time since they became capable of producing viral particles – is an important aspect of the system state for determining the rate of production of Mmod. We are therefore interested in P1 mod (t,a), where a is age, and we must use a partial differential equation to keep track of this state variable. The boundary condition for the PDE accounts for P1 mod(t,0), the production of P1 mod through flipping of the switch in P1 ori cells as well as direct infection by M1 mod.

Production of Mori by senders occurs through intact helper plasmid at rate p

The rate of production of Mmod by P1 mod cells requires consideration of the age of the cells. The rate of production of Mmod by cells of a given age is defined by the function.

where viral particle production by a P1 mod cell begins at age j1 and increases with age to maximal production rate bp at age j2, where 0 < b < 1. Production in these cells is likely slower than the rate in senders, p, because one of the M13 cistrons has been picked apart to allow complementation. The production rate of Mmod at time t is determined from the distribution of P1 mod cells over all ages and the rate of particle production at each age through a convolution:

Analysis

Analysis Analysis Analysis Analysis Analysis Analysis Analysis Analysis

Discussion

DiscussionDiscussionDiscussionDiscussionDiscussionDiscussion

Estimation of Parameter Values

Estimation of Parameter ValuesEstimation of Parameter ValuesEstimation of Parameter ValuesEstimation of Parameter ValuesEstimation of Parameter ValuesEstimation of Parameter ValuesEstimation of Parameter Values

In the absence of precise parameter values, a literature review provided reasonable ranges for the parameters that allowed us to make qualitative statements about the behavior of the system.

• Carrying capacity c was estimated as 1.5*109 cells/ml [1].

• Maximal growth rate g was estimated as ln(2)/20 – ln(2)/30 / min, for a doubling time between 20 and 30 minutes.

• Maximal growth rate of cells producing viral particles was taken as 1/4g – 1/2g [4].

• Adsorption rate k of viral particles to cells was taken as 3*10-11 ml/min [5].

• The rate of flipping of an invertible promoter switch inside a P1 ori cell when induced, S(1), was taken between 0.5 and 1 flips per cell per minute. The rate of switch flipping when uninduced, S(0), was taken between 0 and 0.5 flips per cell per minute.

• The rate of viral particle production p from the intact helper plasmid was taken to be 33 particles per cell per minute [6].

• The rate of viral particle production through complemented helper plasmid with gene VIII knockout is taken as bp, where p is the rate of production from intact helper plasmid and b is between 0 and 1. It is assumed that picking apart the M13 genome for purposes of complementation will either reduce efficiency or have no effect.

• The age at which viral particle production begins in P1 mod cells, j1, and the age at which viral particle production reaches a maximum are assumed to be similar to that of wildtype M13, and these values are taken to be 10 minutes and 50 minutes respectively [6]

• j1 = age at which viral particle production begins in P1 mod cells. Production starts at zero at age j1 and increases.

• j2 = age at which viral particle production

Some interesting questions that we can pose in the model could be what would happen if the parameters that were chosen were allowed to vary significantly. These questions could be answered by analyzing our model with a few changes. Some questions could be

- What would happen if the reduction in efficiency, r, would change to different factors? Even if the system has already been induced.

- What initial conditions should we start with so we can attain the values we hoped for?

- What is the threshold of leakiness should we allow and how would the model break if the leaky promoter were quite severe?

We shall now examine the first question.

Below, we have values of the reduction in efficiency plotted for various values of r. Namely, we have r = 0.01, 0.1, 0.25, 0.75 and 1. We would also like to note that these graphs were plotted assuming that the inducer has been put in, s=0.5, and the initial conditions of the bacteria is a 1:10:5 where the first value is the sender population, the second value is the primary receiver population and the third value is the secondary receivers.

As we can see, as r increases, the secondary modified message would increase which would make sense since as the production of the secondary phagemid is more efficient, it would create more viruses thus there would be a greater volume infecting the cells. There is one remark that is interesting and that is of when r = 0.01 and that is even when the inducer has we induced into the environment, there are not enough virus to infect the secondary receivers. We would also like to note that when the secondary phagemid is not efficient, the primary virus is infecting all the secondary cells. Now we will examine the second question. Below, would be the graphs of different ratios of the starting populations in an induced environment with the reduction of efficiency set to 0.5.

As shown, when the initial conditions are at a ratio of 1:20:10, we see this seems to be the most optimal since this is in the induced system; we have a strong presence of the secondary modified message. This is a strong statement because it shows that the secondary phagemid is indeed being produced by the primary cells.

For these two situations when the primary population is too low, there are not enough primary cells to be infected to produce the secondary phagemid thus there would be no modified secondary population. The reason why the secondary population with the original is so high is because the sender phagemid have nothing else to infect thus must infect the secondary cell population.

For the graphs above, we see that the secondary cells is the least thus there are not enough of the secondary cells to be infected. For the top graph, there is also a lot of secondary cell with original message because the primary cells get infected first thus would have more time to multiply which would then get infected by the primary phagemid since there are an abundance of them. For the bottom graph, we see that the primary cells are absorbing all the sender phagemid and due to the scarcity of the secondary cells, are not being infected with the secondary phagemid that would explain the low levels of the secondary cells infected with the secondary phagemid.

In this case, we see that there is a large amount of secondary cells infected with the secondary phagemid. This is due to the high amounts of secondary cells available for the secondary phagemid to infect. The small concentration of primary infected cells is due to the low amount of sender population that is infecting the primary cells however when the secondary phage is produced, it would quickly infect a secondary cell. Now we shall analyze the third question An interesting question would be how much is leakiness involved with the model and what would be the upper bound on leakiness that would conform to the model. Similar to the previous graphs, the initial conditions are a 1:10:5 ratio and the reduction of efficiency is 0.5.

As we can see when the probability of leakiness is on a magnitude of 10^-3, it is well behave as in the secondary modified cells are not as present since there are not a lot of secondary phagemid to infect the cells. However, as shown above, when the magnitude is about 10^-2, it seems as though there are more secondary phagemids that can infect the secondary cells which would cause a large concentration of the secondary modified cell. Thus as we have seen in this analysis that there are some questions that can be solve however many questions can be asked since there may be improvements to the model or the initial parameters themselves.


 References:

3. Short protocols in molecular biology, Fred Ausubel et al., 5th ed. Vol. 1 pp. 1-5

4. Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbour, NY. 2001.

5. Tzagoloff, H., and D. Pratt. 1964. The initial steps in infection with coliphage M13. Virology 24:372?380.

6. Clackson, T., & Lowman, H. B. (2006). Phage display, a practical approach. (pp. 2-14). New York, NY: Oxford University Press, USA.

Phage Particle Production Modelling

'''Description''' M13’s relatively small genome can be classified in three succinct subsets: structural (genes III, and VI - IX), morphogenetic (genes I, IV, and XI), and replicative (genes II, V, and X). The replicative genes play the largest role in our project, but in order to model the behaviour of the virus from infection to secretion, we must take into account all of the genes, their respective proteins, and their functions. By creating a differential model of the set of mRNA strands, proteins, and DNA forms present inside a bacterial host cell at any point in time, one can see the effects of increasing or decreasing levels of specific compounds on viral production and packaging. The main function behind each of the proteins can be found in the main project summary. The protein produced by the translation of gene V (pV) is responsible for much of the regulation of viral replication, and thus is central to the model. If there is too much pV present in the cell, it will sequester the infected form (IF) DNA and stop the replicative process; too little, and the process can’t take place [3-7]. As the concentration of pV stabilizes (along with the other regulatory proteins, pII, pV, and pX), a steady state of viral production and secretion is achieved. The model constructed by the viral assembly team is complimentary to the goals of the switch and the population dynamics teams. The designs conceived by the switch team could be mathematically tested by the assembly model. For example, what is the effect of removing a subset of genes from M13’s genome? Or, will assembly/secretion still occur if those genes are re-introduced after a switch event? Further, the fidelity of the population dynamics model would benefit from having realistic estimates of viral secretion rates over time. This is a specific quantity the assembly model hopes to determine. The assembly group’s research and model development has implications on the project’s design. Through the model, genes that were mandatory for assembly or secretion can be characterized. Selecting which genes to withhold from the primary receivers (i.e. which genes to include on the helper phagemid) relies on this info. The wrong choice could result in host cell death or crippling of the infecting viral particles, preventing retransmission (the goal of the design). Testing such choices with a mathematical model rather than in a lab experiment saves both time and money. Citations: [1] Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbour, NY. 2001 [2] Baas, P. D. (1985). Biochim. Biophys. Acta. 825, 111-139 [3] Mazur, B. J. & Model, P. (1973). J. Mol. Biol. 78, 285-300. [4] Webster, R. E. & Cashman, J. S. (1973). Virology. 55, 20-38. [5] Mazur, B. J. & Zinder, N. D. (1975). Virology. 68, 490-502. [6] Geider, K. & Kornberg, A. (1974). J. Biol. Chem. 249, 3999-4005. [7] Salstrom, J. S. & Pratt, D. (1971). J. Mol. Biol. 61, 489-501. [8] Fulford, W. "Bacteriophage F1 DNA Replication Genes *1II. The Roles of Gene V Protein and Gene II Protein in Complementary Strand Synthesis." Journal of Molecular Biology 203.1 (1988): 39-48. Print. [9] Haigh, Nora G., and Robert E. Webster. "The pI and pXI Assembly Proteins Serve Separate and Essential Roles in Filamentous Phage Assembly." Journal of Molecular Biology 293 (1999): 1017–1027. [10] Russel, Marjorie. "Interchangeability of Related Proteins and Autonomy of Function: The Morphogenetic Proteins of Filamentous Phage f1 and IKe Cannot Replace One Another." Journal of Molecular Biology 227 (1992): 453-462. [11] Wickner, William, Gail Mandel, Craig Zwizinski, Marjorie Bates, and Teresa Killick. "Synthesis of phage M13 coat protein and its assembly into membranes in vitro." Proceedings of the National Academy of Sciences 75.4 (1978): 1754-1758. '''Equations''' \begin{equation} \frac{d[S_j]}{dt} = \alpha_j[RF] - \delta_j[S_j] \end{equation} \begin{equation} \frac{d[P_i]}{dt} = \beta_i[M_i^F] - \delta_D[P_i] \end{equation} \begin{equation} [M_i] = \sum_{\text{mRNA}}[S_j] \end{equation} \begin{equation} [M_i^F] = \left(1 - \frac{[P_5]}{k_i + [P_5]}\right)[M_i] \end{equation} \begin{equation} \frac{d[RF]}{dt} = k_{conv}[IF^F] - \delta_D[RF] \end{equation} \begin{equation} \frac{d[IF]}{dt} = k_{RC}\left(1 - \frac{[P_2]}{H + [P_2]}\right)[RF] - k_{exp}[IF^S] - \delta_D[IF] \end{equation} \begin{equation} [IF^F] = \left(1 - \frac{[P_5]^n}{K^n + [P_5]^n}\right)[IF] \end{equation} \begin{equation} [IF^S] = \left(\frac{[P_5]^n}{K^n + [P_5]^n}\right)[IF] \end{equation}\\* \begin{equation} \forall m \in \{1, \dots, 8\},\;\; \forall j \in \{1, \dots, 11\} \end{equation} '''Variables''' S_j : The jth mRNA sequence, with j starting at the gene 2 promoter α_j : Transcription rate from of jth promoter β_i : Translation rate from gene i mRNA δ_j : Degradation rate of jth mRNA δ_D : Dilution rate k_conv : Rate constant for IF → RF converstion k_RC : Rate constant for rolling-circle replication k_exp : Rate constant for export of sequestered IF from infected cell k_i : Half-saturating constant for P5 binding to mRNA for gene i H: Half-saturating constant for P2 binding to RF K : Half-saturating constant for P5 co-operative binding to IF n : Co-operativity constant for P5 binding to IF M_i : mRNA coding for the ith gene on the M13 genome, as labelled in literature M_i^F: Unbound (“free”) mRNA for gene i P_i : The protein product of the ith gene RF: “Replicative Form” (double stranded) viral DNA IF: “Infective Form” (single-stranded) viral DNA '''Output''' '''Discussion''' Our governing differential equations reflect our attempt to model M13’s genetic regulatory mechanisms with appreciable fidelity. All direct byproducts of the viral genome were regarded as state variables over time, and degradation/dilution play a role in each synthesis equation. Equations (20) and (21) correspond to mRNA and subsequent protein synthesis, with considerations for unique translation and transcription rates, captured by our estimated parameters. Equation (22) accounts for the fact that mRNA concentrations for a specific gene are given by the sum of all pertinent mRNA chains (chains which contain said mRNA), as described by the known order of genes and promoters in the genome. In equation (23), P5 plays the role of regulating the translation of all mRNA. This is critical; by hindering its own synthesis, it creates a stabilizing negative feedback loop. Equations (24) through (27) measure the rates of change of each form of viral DNA. RF synthesis (equation (24)) is controlled by host processes and depends on free IF DNA. Equation (25), which considers total IF DNA synthesis, acknowledges the role of P2 in rolling circle DNA production, as well as the fact that IF DNA is constantly being sequestered by P5. Free IF DNA and its counterpart, sequestered IF DNA, have rates of synthesis that depend on the total IF concentration and incorporate co-operativity, as suggested by previous M13 research. Through its presence in equations (23), (26), and (27), P5 asserts its role as a key player in the genetic regulatory system.

The University of Waterloo’s iGEM – Human Practices team is a diverse team whose goal is to raise awareness on issues regarding synthetic biology. The team also provides the student community information about the latest in the research area of synthetic biology to help the community make informed, accurate and fact-based opinions. Our goal is to strengthen the bridge between the community and their knowledge of synthetic biology while eliminating misconceptions regarding synthetic biology.

In the past year, the team gained valuable experience and information through the projects they worked on. Each project provided more insight on how informed the student community is on the topic of synthetic biology. This further helped us plan out activities that helped us achieve our goal.

One of the main purposes behind the projects this year to enrich, educate and empower the student community. To achieve this goal, various activities were planned to inform the student community about the field of synthetic biology, it’s potentials and how it affects the world around us. These activities provide fundamental knowledge of synthetic biology and it’s uses, allowing the participants and the viewers to form an informed, accurate and fact-based opinion about the topic.

T.I.L. Syn Bio

iGEM is a community of people passionate about synthetic biology – how can we best convey this while reaching out to the student community? Sometimes reading papers and textbooks doesn't quite do it for understanding an idea. As students, we know it can be difficult to grasp some concepts we’re not familiar with. So what’s a better way to communicate an idea? Could social media be the answer? That was the idea behind the VLOG series TIL: Syn Bio.

These series are a quick and effective way to convey the ideas and passion of synthetic biology. The series has many episodes that highlight various aspects of synthetic biology through a mixture of one-on-one videos and animated style videos. The series begin with episodes explaining “What is Synthetic Biology?, “Fundamental Advances” and “Cell-to-Cell Communication” (Waterloo iGEM’s 2013 project). This phase of the series is important to orient the viewers and provide some background information.

igem-cmit from Waterloo iGEM on Vimeo.

During the second part of the series, the team takes a fun twist. Using the TIL: Synthetic Biology outreach event footage to compare the viewpoints of students and professors on various topics relating to synthetic biology. The footage from this event is used for addressing many factors associated with the idea of synthetic biology. These factors range from the background knowledge to stigma associated with synthetic biology and from the regulations needed to its future potential.

The series begins with these six videos, leaving the rest of the series to be shaped by viewers. Ultimately, viewers engage with the team about what they want to see in future videos, ask questions they want answered and connect with information from a variety of sources.

The TIL: Synthetic Biology outreach event (used as part of the video series) was well-received. The team came prepared with questions to ask passing students. Students were also given 4-5 days notice via Facebook. The idea behind this aspect of the video was to have it be a surprise. Questions like "do you support GMOs?", "would you eat modified fruit/meat?", "who should be able to practice synthetic biology/should it be open sourced?" and many more were asked. The team was in for some surprises with the diversity of knowledge on campus! We hope incorporating the footage into our series will give participants a fun look into their experience, which they can easily share with their friends and family. Overall, we hope that the team's work will inspire more leaders to take part and contribute to the advances in synthetic biology, regardless of their academic or professional background.

Intent to Invent

Intent to Invent was hosted on March 07, 2013 at the University of Waterloo’s Quantum Nano Center. The purpose of the event was to:

1. Connect the students to experts in 3 key industries that use synthetic biology in their processes: Agriculture, Health and Pharmaceuticals.
2. Bridge the level of discomfort a scientist has in regards to business.
3. To encourage entrepreneurship within the scientific community by delivering resourceful content from industry experts.

The event promoted open panel discussions of emerging technologies in biotechnology and other advanced biological fields within the 3 industries. Students got a chance to see how synthetic biology is the connected to entrepreneurship, innovation and commercialization. They learned about the industry perspectives and barriers faced by biological companies at different stages in their business model. This talk also encouraged entrepreneurship within the scientific community by delivering resourceful content form industry experts. Each speaker gave a 20-minute mini lecture on topics including: Clinical Trial Drug Development, Commercialization of Biomass and Energy Products and Entrepreneurial Barriers for Biotechnology Companies.

Steve jobs once said, “I think the biggest innovation of 21st century will be the intersection of biology and technology. A new era is beginning, just like the digital one…”. Through sessions such as Intent to Invent, Waterloo iGEM hopes to enrich the experience of science enthusiasts as well as those just curious about synthetic biology and it’s potential. By connecting these students to industry experts, we were able to gage their interests in an innovative and entrepreneurial aspect of science. Many students showed interest in learning more about the bridge between science and business in the future. iGEM received good feedback regarding Intent to Invent, as many students felt that the information they learned was very valuable. Waterloo iGEM provided many students the appropriate connection and information they need to start connecting the scientist in them with the businessman/businesswoman in them.

VeloCity Science

How do we inspire young people to eliminate the gap between science and business? The conventional education system does not provide for this overlap. There is a job unemployment crisis throughout North America, and from the past experience, this is the perfect time to turn to entrepreneurship for solutions. It is time to do what we have done to the IT industry back in the 80s, but with biotechnology this time. The perfect storm is brewing. Economic downturns have proven to be the best time for entrepreneurship. The Canadian government has seen this and supports entrepreneurial initiatives like these. And most importantly, we have bright young people hungry to make changes to the world.

And that’s why VeloCity Science has been started, an entrepreneurship program that brings together the right business resources (networks, mentorship, legal and financial services, etc.) and the right technical resources (wet-lab space, consumables and equipment) to create kick-ass biotechnology start-ups.

But that’s not all. Above of all these resources, it is the sense of community that is crucial for the success of these entrepreneurs. The University of Waterloo has proven time and time over in providing a strong sense of community to our entrepreneurs through the programs like Accelerator Centre, Communitech Hub, and VeloCity.

That’s the story of VeloCity Science, and we are just starting to write it.

Laboratory

Intent to Invent

Safety

All experiments are carried out in a BL2 certified lab. Researcher safety when using E. coli, would not be compromised in safety issues due to use of M13. It poses no threat at all to humans. While, the E. coli strain used was relatively harmless, treatment of possible infections may potentially be affected by the antibiotic resistance. Furthermore, spontaneous mutations which result in increased infectivity may result. However, measures and precautions suggested by the Canadian biosafety guidelines were taken to minimize even the slight chance of infection. Additionally, the working conditions of the lab is already above the recommended safety level of BL1 for usage of M13 viruses. Every member of the team has been trained with safety modules and went through a week of lab training and continuous oversight from the Advisors and his graduate students in the lab. All lab members, including graduate students or other students that were working in the lab, wore appropriate PPEs and disposed all consumable in appropriate biological waste boxes. All surfaces were wiped down with ethanol after use and all glassware was washed immediately after their usage.

The design of the project does not call for release into the public. Additionally, the project design does not produce any harmful products. Through it is possible that the construct could get released to the general public accidentally. But, the product of the constructs only produce fluorescent proteins and it can only be used in a controlled setting with a certain type of chemical present in the environment, thus making it ineffective when released to the public. Because safety of the public and the lab members is our utmost concern, we have ensured that all wastes are thrown out appropriately and autoclaved so that accidentally release would never occur.

There are no additional risks posed by our projects compared to other general BL1 lab concerns. Our bacteria are not pathogenic and are unable to survive outside of the lab environment, because they are unable to effectively compete with other organisms in nature. As stated above, all wastes are discarded according to the Waterloo standards and autoclaved.

There is no potential for harm to human health through use of our constructs, as described above. There is therefore no risk of malicious use.

Our constructs pose no threats to human health, as described above, and scaling up would not change this. Our project is a "fundamental advance" that contributes to the coordination of population-level cellular behavior by allowing messages to be sent between populations of E. coli cells. However, many additional layers of complexity in engineering would be required to use our method to enable pathogenic or otherwise dangerous behaviors in populations of cells.

The cell to cell communication project does include packaging viral particles. Although there are only some proteins of the M13 virus that are packaged and are therefore not a safety risk. M13 is not a safety risk even if its whole genome is packaged. Our project poses no threat to safety and thus we haven't implemented any of these mechanisms.

All the lab and design team members successfully passed the following safety training: Employee Orientation Training Session: https://info.uwaterloo.ca/infohs/hse/online_training/employee-orientation/Staff%20Orientation.swf Workplace Violence and Harassment Training: https://info.uwaterloo.ca/infohs/hse/online_training/workplace_violence/workplace_violence.html General Laboratory Safety: https://info.uwaterloo.ca/infohs/hse/online_training/lab_safety/lab_safety_course.html WHMIS: http://www.safetyoffice.uwaterloo.ca/hse/lab_safety/index.html Laboratory BioSafety Training: https://info.uwaterloo.ca/infohs/hse/online_training/biosafety/biosafety.swf

The BioSafety Guidelines followed by uWaterloo iGEM team can be found here: http://www.safetyoffice.uwaterloo.ca/hse/bio_safety/legislation.html

University of Waterloo has a Biosafety Committee and can be found here: http://www.safetyoffice.uwaterloo.ca/hse/bio_safety/bsc.html. Although the project has not been discussed with the Biosafety Committee, it has been discussed with several faculty members and has been found to have no risks. Furthermore, the laboratories operating at the University of Waterloo have obtained permits from the Bio-Safety Committee in order to perform intended research. Since the Waterloo iGEM team performs all laboratory work in a parent lab under the guidance of the Masters and PhD students of that lab, the permits obtained by the parent lab cover the projects carried out in the lab.

Canada has very well established biosafety regulations and guidelines which can be found here: http://www.phac-aspc.gc.ca/lab-bio/

The laboratory we work on cell to cell communication project is rated level 1.

E.coli strains that Waterloo iGEM team works with falls within the risk level 1. Additionally the laboratory we operate in is certified for work with the above listed risk group of the E.coli.

Administrators

Lab & Design

Team Leaders

Team

Mathematical Modelling

Human Practices

Advisors

Graduate Student Advisors

Web Developer

Acknowledgements

We would like to offer special thanks to the following groups for your help

• Our advisors for donating your time and intellectual knowledge to the team

• Members of the Charles lab from University of Waterloo for your generous sharing of lab space, equipment and constructive suggestions to our project.

• Endy lab from Stanford University for proposing the use of cell-cell DNA messaging and sharing of knowledge and parts.

• Monica Martinez from Endy lab for your supportive role in our project and expertise in the usage of M13.

• Our IGEM collaborator this year: team UOttawa for helping us with the making of our constructs and interchange of knowledge.

• Staff of University of Waterloo Department of Biology for your timeless support and encouragement to our team.

• Dr. Maud Gorbet's from University of Waterloo for the sharing and expertise on flow cytometry for detecting reporter molecules.

• Dr. Mongol Marsden from University of Waterloo for the use of fluorescent microscope

• Susanna Zucca from Magni lab of Università degli Studi di Pavia for your sharing of parts for the single copy switch experiment.

• Andrew Dhawan for your guidance for the Mathematical Modelling team.

• Dr. Roderick Slavcev from pharmacy for his guidance with M13 bacteriophage.

• Dr. Gord Surgeoner, Dr. Catherine Burns, and Nicky Arvanitis for your insight presentation at our Intent to Invent seminar.

• All members of University of Waterloo IGEM 2013 for your sleepless nights and love for synthetic biology