Team:FCB-UANL/Description
BIO-INSPIRED SOLUTION
OBJECTIVES
CIRCUITS
Ranaspumins
Surfactin
Biofilm
THE FOAM
VERSATILITY
BIOCONTAINMENT
Killswitch
REFERENCES
FIRE INCIDENTS, WHAT IS THE REAL PROBLEM?
In recent years, news about fire incidents have become more and more
frequent. They are particularly alarming since they endanger both human
lives and our environment, in addition to occurring in increasingly wide
geographic regions, greatly complicating their combat. Given their
importance, more and more strategies and tools are used to combat them.
However,what if the solution is not really a solution?
Although it may seem controversial to say that fighting fires is a problem, the reasons stem from the lack of efficient methods to combat them, which have two main consequences: First, the slow solution results in a prolonged existence of the fire, along with the long-term harmful effects of the few efficient combat tools available, which are typically firefighting foams.
While the use of water is another common combat tool used, it is not very effective in all fire types. Complicated with the fact that it is inconceivable to use hundreds of gallons of the vital fluid within one of the states with more prolonged periods of drought and a growing water crisis in Mexico (further information of the social component of this problem is provided in our human practices section). On the other hand, foams are expensive and difficult to obtain, since there are no Mexican producers and some of the current producers are not offered in the Mexican market (to get to know more, go to our entrepreneurship section).
Although it may seem controversial to say that fighting fires is a problem, the reasons stem from the lack of efficient methods to combat them, which have two main consequences: First, the slow solution results in a prolonged existence of the fire, along with the long-term harmful effects of the few efficient combat tools available, which are typically firefighting foams.
While the use of water is another common combat tool used, it is not very effective in all fire types. Complicated with the fact that it is inconceivable to use hundreds of gallons of the vital fluid within one of the states with more prolonged periods of drought and a growing water crisis in Mexico (further information of the social component of this problem is provided in our human practices section). On the other hand, foams are expensive and difficult to obtain, since there are no Mexican producers and some of the current producers are not offered in the Mexican market (to get to know more, go to our entrepreneurship section).
A BIO-INSPIRED SOLUTION
Analyzing this situation, we decided to develop the first ever
synthetic biology based firefighting foam for type A and B
fires. For us to achieve this, we got inspired by the most
efficient foaming compounds found in nature: the Ranaspumin proteins
that come from a local frog species. Since we are a two-year project, we
started on the basis settled by the FCB-UANL 2020 (1) team with the
proper genetic circuit designs (explained later in this
section).
With our project, we aim to contribute to accomplish the Sustainable Development Goals (SDGs) adopted by the United Nations in 2015. We aim to accomplish the SDGs 9: industry, innovation and infrastructure, by generating a bioentrepreneurship proposal that promotes the biotechnology industry in our region; 15: life on land, by generating an alternative for the contaminant compounds that every year are released into the ecosystem; and 17: partnerships for the goals, by collaborating with a lot of people both inside and outside of the iGEM community.
Our project is the first of its kind, since it is the first time ever a team works with this issue and develops a firefighting foam. Building something never seen before on iGEM represents a big challenge. For that, we are excited to present our overall objectives for the iGEM season.
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With our project, we aim to contribute to accomplish the Sustainable Development Goals (SDGs) adopted by the United Nations in 2015. We aim to accomplish the SDGs 9: industry, innovation and infrastructure, by generating a bioentrepreneurship proposal that promotes the biotechnology industry in our region; 15: life on land, by generating an alternative for the contaminant compounds that every year are released into the ecosystem; and 17: partnerships for the goals, by collaborating with a lot of people both inside and outside of the iGEM community.
Our project is the first of its kind, since it is the first time ever a team works with this issue and develops a firefighting foam. Building something never seen before on iGEM represents a big challenge. For that, we are excited to present our overall objectives for the iGEM season.
>
PROJECT OBJECTIVES
For us to complete our project development, we
established several objectives enlisted below:
- Characterize the genetic parts of the Ranaspumin proteins (the core of our foam), optimizing their production with both experimental data and model predictions (for more information go to our engineering success section)
- Validate the expression model developed by team FCB-UANL 2020 (1), and develop new models to further complement the data (for more information go to our model section)
- Produce our Ranaspumin proteins and characterize their foaming properties by carrying out foam tests
- Continuously validate the development of our project by establishing communication with stakeholders (explained in our human practices section)
- Make a thorough business plan to evaluate the feasibility of our project as a product for the Mexican market (reported in our entrepreneurship section)
- Analyze the complete legal framework, as well as the biosafety and biosecurity aspects revolving our project (a detailed description is provided in our safety section )
- Gather all the information obtained to develop our proposed implementation: the way our product would be used to actually solve the problem we are working with (further information in our proposed implementation section )
- Communicate our project to the society in a proper way, using the adequate pedagogical and communicational tools (further information is provided in our communication , education , and inclusivity sections)
GENETIC CIRCUITS
As previously explained, the genetic circuits used for the
production of our foam were designed last year by the iGEM FCB-UANL
2020 team (1). Next, we present the circuits and a brief description
of them. For further information of the decision-making process
please visit the previous work done by the team FCB-UANL 2020 (1).
In addition, some alternative components for our foam are explained
later in this section.
RANASPUMIN PRODUCTION
All the Ranaspumins proteins will be produced on E. coli. We plan to
separately produce Rsn-2 (BBa_K3498009 ) and Rsn-3-5 (BBa_K3498010 , BBa_K3498011 , BBa_K3498012 ) using two different strains; however, both are regulated by a
vanillic acid inducible promoter (pVanCC) (BBa_K3317006 ), designed RBS (using the tool RBS calculator), and the
terminator rrnbT1 (BBa_B0010). In
addition, it is planned to constitutively express VanR AM (BBa_K3317004
) (the regulator of pVanCC) using the constitutive promoter
PlacIQ (BBa_K3498001 ) and van2 RBS (BBa_K3498014 ), as recommended by Meyer and collaborators (2).
The next image shows the circuits designed for Rsn-2 (BBa_K3498029 ) and Rsn-3-5 (BBa_K3498030 ) production, in which the constitutive expression of the regulator (BBa_K3498031 ), as well as the regulable expression of Rsn-2 and Rsn-3-5 respectively is included.
The next image shows the circuits designed for Rsn-2 (BBa_K3498029 ) and Rsn-3-5 (BBa_K3498030 ) production, in which the constitutive expression of the regulator (BBa_K3498031 ), as well as the regulable expression of Rsn-2 and Rsn-3-5 respectively is included.
SURFACTIN PRODUCTION
Next, we used the circuit (BBa_K3498025
) for the production of surfactin, our secondary foaming agent.
Due to the complex regulatory network of surfactin production, this
circuit activates the SrfA operon (in charge of surfactin
production) by an indirect regulatory system. Through the
overexpression of PhrC (BBa_K3498002 ), it is planned to inhibit the RapC protein, since it is a
negative regulator of ComA, the direct activator of SrfA. Hence, it
is a double negative regulation, where the inhibitor of the
activator of surfactin production is the main target. The circuit
also contains sfp (BBa_K3498005 ), a gene coding for an activator of the surfactin synthetase
enzyme, as well as AbrB (BBa_K3498006 ) and Spo0E (BBa_K3498003 ) sporulation delayers.
PhrC and sfp overexpression are regulated by a xylose-inducible promoter (BBa_K823015) (which regulator, XylR, will be constitutively expressed), as well as AbrB using P43 promoter (BBa_K143013). Next, Spo0E (expression will be regulated through the presence of Spo0F (BBa_K3498000 ) (part of the sporulation pathway). The circuit also includes cat (BBa_J31005 : a chloramphenicol resistance gene), and the homologies AmyE-5’ and AmyE-3’ for genome integration (BBa_K143001 ).
PhrC and sfp overexpression are regulated by a xylose-inducible promoter (BBa_K823015) (which regulator, XylR, will be constitutively expressed), as well as AbrB using P43 promoter (BBa_K143013). Next, Spo0E (expression will be regulated through the presence of Spo0F (BBa_K3498000 ) (part of the sporulation pathway). The circuit also includes cat (BBa_J31005 : a chloramphenicol resistance gene), and the homologies AmyE-5’ and AmyE-3’ for genome integration (BBa_K143001 ).
BIOFILM PRODUCTION
For the biofilm production (BBa_K3498027
), the regulatory mechanism pays special attention to the eps
and tapA operons. Just as in the surfactin production, indirect
regulatory mechanisms are used; on this case, the circuit aims the
overexpression of SinI (BBa_K3498007
) and AbbA (BBa_K3498006
), inhibitors of SinR and AbrB respectively, which inhibit
biofilm formation.
SinI and Abba genes are regulated by the xylose-inducible promoter (which regulator, XylR, will be constitutively expressed). All the parts used (Pxyl promoter, Spo0E , Spo0F promoter, cat, and even the Amy homologies) are the same reported for the surfactin circuit.
SinI and Abba genes are regulated by the xylose-inducible promoter (which regulator, XylR, will be constitutively expressed). All the parts used (Pxyl promoter, Spo0E , Spo0F promoter, cat, and even the Amy homologies) are the same reported for the surfactin circuit.
SYNBIOFOAM COMPOSITION
Since our proposed components have never been used for foam
production, we decided to do some research regarding their
physicochemical properties, in order to be sure we acceptably
selected them as foaming and stabilizing agents. For this purpose,
we decided to take into account three main aspects, [1] the half
life, [2] their surfactant properties (this is, their ability to
lower the water’s surface tension, (3), as it facilitates bubble
formation (4), and [3] their foamability. Next, we explain some of
their properties.
RANASPUMINS
Half-life: Foam formed by Ranaspumins in a natural environment
has a varied half-life that can go from days to weeks, even under
arduous tropical conditions at 1-2 mg/mL concentrations. However,
Rsn-2 alone tends to have a shorter half-life (4).
Surfactant properties: They have been reported to be efficient surfactants, due to their uncommon amino acid sequence, which has a highly charged hydrophilic portion, along with an appreciably non-polar N-terminal sequence. These amphiphilic characteristics are on a much larger molecular scale than those from common surfactants (4).
Natural foam fluid can "wet" hydrophobic surfaces, reducing surface tension at total protein concentrations as low as 10-100 µg/mL. Therefore, it is more effective than proteins such as lysozyme or bovine serum albumin (BSA) (4).
Foamability: The most well-characterized protein of the family, Rsn-2, can achieve higher foamability than Tween 20, Triton X100, or BSA. It is able to form up to double the volume of foam from a given amount of liquid (5).
Surfactant properties: They have been reported to be efficient surfactants, due to their uncommon amino acid sequence, which has a highly charged hydrophilic portion, along with an appreciably non-polar N-terminal sequence. These amphiphilic characteristics are on a much larger molecular scale than those from common surfactants (4).
Natural foam fluid can "wet" hydrophobic surfaces, reducing surface tension at total protein concentrations as low as 10-100 µg/mL. Therefore, it is more effective than proteins such as lysozyme or bovine serum albumin (BSA) (4).
Foamability: The most well-characterized protein of the family, Rsn-2, can achieve higher foamability than Tween 20, Triton X100, or BSA. It is able to form up to double the volume of foam from a given amount of liquid (5).
SURFACTIN
Half-life: Previous reports of storage of 4°C for over 2
months and in a frozen state for more than a half year did not
likewise impact the concentration of the surfactin (6). On the
contrary, in a foam state it has a half-life of 141 seconds at
concentrations of 0.2 mg/mL (7). However, it has not been proven on
foams with other compounds.
Surfactant properties: It is able to reduce water surface tension from 72 Nm/m to 27.90 mN/m, even at concentration of 0.005% (8), being the most powerful natural surfactant known (3). It can present little differences among strains, and the varieties have shown the same properties even after drying out by splash drying and reconstitution, and being exposed to the high temperatures of autoclaving (9).
Foamability: Even though their foaming properties have not been well studied, it was found that it is actually able to form foams (7). Also, in the reports of their up-scaling process there are several reports of foam formation due to the bioreactor agitation (3).
Surfactant properties: It is able to reduce water surface tension from 72 Nm/m to 27.90 mN/m, even at concentration of 0.005% (8), being the most powerful natural surfactant known (3). It can present little differences among strains, and the varieties have shown the same properties even after drying out by splash drying and reconstitution, and being exposed to the high temperatures of autoclaving (9).
Foamability: Even though their foaming properties have not been well studied, it was found that it is actually able to form foams (7). Also, in the reports of their up-scaling process there are several reports of foam formation due to the bioreactor agitation (3).
BIOFILM
Half-life: While there is no exact data, its long duration is
known (10). In addition, it has exhibited resistance to wetting and
gas penetration, considered as extremely nonwetting (11).
Surfactant properties: Since biofilm is a complex matrix (11). It is not thought to be used as a surfactant, but as a stabilizer, making our foam more stable by avoiding water and oxygen to pass.
Foamability: It surpasses the repellency of Teflon toward water and lower surface tension liquids, as well as preventing the passage of gases and vapors (11). These mentioned characteristics, being able to block oxygen from entering the combustion reaction and thereby acting as a film, makes the biofilm an excellent firefighting foam component.
Surfactant properties: Since biofilm is a complex matrix (11). It is not thought to be used as a surfactant, but as a stabilizer, making our foam more stable by avoiding water and oxygen to pass.
Foamability: It surpasses the repellency of Teflon toward water and lower surface tension liquids, as well as preventing the passage of gases and vapors (11). These mentioned characteristics, being able to block oxygen from entering the combustion reaction and thereby acting as a film, makes the biofilm an excellent firefighting foam component.
VERSATILITY: CHANGING THE FOAM’S COMPOSITION
As reported in our human
practices section and experienced in our lab work reported
in the engineering
success section , there are different types of fire, and
each one of them have different requirements due to factors such as
the temperature, fuel, etc. Even though our main goal is to create
an AB type firefighting foam, we are aware of the necessities of our
community and the importance of having different alternatives to
combat different fire types.
Hence, inspired by the synthetic biology principles, we have decided to use modularity on our foam composition as well. Next, we provide a list of alternative components to be added in our foam, as a future plan to extend our product varieties. Next, we present the bio-inspired components we have considered as potential foam components.
Hence, inspired by the synthetic biology principles, we have decided to use modularity on our foam composition as well. Next, we provide a list of alternative components to be added in our foam, as a future plan to extend our product varieties. Next, we present the bio-inspired components we have considered as potential foam components.
ALTERNATIVE COMPONENTS
As mentioned above, we have selected the following components based
on their properties for foam formation or stabilization. However, we
are aware that before trying to add them to a variety of synbiofoam,
a complete risk analysis has to be carried out.
| Component | Source | Description | Foam Function |
|---|---|---|---|
| Environmentally friendly products found in nature | Expensive research & development process | Migration toward environmentally-friendly society | Difficulty competing with chemical surfactants |
| Personalized customer service | Use of other equipment for industrial production | Gradual increase in biotechnology industry | Already established companies with greater capital power |
| Beneficial use of wastes from another industry | New legislations favoring less-harmful chemicals | Additional regulations to deal with when working with microorganisms and products derived from biotechnology techniques. | |
| Latherin | Equus caballus (horse) | Approximately 1 mg/mL of this element substantially decreases water surface tension on a hydrophobic surface, being more efficient than lysozyme or BSA. | May be an additional stabilizer for film formation, as well as a secondary foaming agent. |
| Ranasmurfin | Litoria spenceri (tree frog) | It is associated with high hydrophobicity and/or increased viscosity of denatured protein, in which physical entrapment of air bubbles is facilitated in concentrated viscous mixtures (4). | May help to give more viscosity to the mixture, as well as increase the amount of foam formed from the same amount of liquid. |
| Rhamnolipids | Pseudomonas aeruginosa | They are able to modify surface tension, solubilization, and motility stimulation. | May primarily aid foaming by increasing the reduction in surface tension. |
| Alkyl Polyglucosides | Different organisms, such as sugar cane | Used to obtain oil due to its emulsifying activity. Also, they can interfere with surface tension and resists high temperatures (12; 13; 14). | May primarily aid in foaming by increasing the reduction in surface tension and increasing thermostability. |
| Albumin | Gallus gallus domesticus (hen) | It is used to stabilize proteins; it coagulates at a temperature of 50°C forming hydrophobic aggregates. In the presence of surfactants, depending on the complex formed, the surfactant properties of the rest of components can be enhanced (15; 16; 17; 18). | It could be evaluated with the objective of incorporating it to a lesser or greater extent, by forming hydrophobic aggregates. |
| Glycolipids | Torulopis bombicola | They have shown a substantial decrease in surface or interfacial tensions, but could not stabilize either hydrocarbon-in-water or vegetable oil-in-water emulsions (19). | May help foam formation to a lesser extent, by slightly increasing the reduction in surface tension. |
| Biosufactant | Rhodococcus (sp.) | It can reduce the surface tension in distilled water from 72.5 to 30.7 mN/m at certain concentrations (20). | May primarily aid foaming by increasing the reduction in surface tension. |
| Biosufactant | Staphylococcus (sp) | It has shown reduction in surface tension, as well as stability in pH from 2 to 12, high temperatures, and in saline environments (21). | May primarily aid foaming by increasing the deduction in surface tension and thermostability. |
INTEGRATED BIOCONTAINMENT CIRCUIT
SHAPING OUR APPROACH
While analyzing the safety risks of our project (further detailed in
our safety
section), we identified one related to working with
sporulating bacteria and recombinant DNA: accidentally releasing
modified spores in the environment.
Spores are a morphological state that give resistance to many survival-threatening conditions, such as nutritional limitation, temperature, radiation, toxic compounds, high pressures, and competition with other microorganisms (22; 23). Evidently, cells in this state are more difficult to remove or kill. The risk of our modified spores unintentional release directly repercutes on the biosafety aspects of our foam.
Last year, the iGEM FCB-UANL 2020 team (1) worked on a system to delay sporulation through the utilization of Spo0E phosphatase, dephosphorylating Spo0A-P which at high concentrations activates sporulation. However, this year we designed a model to force the cell to enter a competence state instead of sporulation. Unfortunately, our model predicted that such a mechanism is difficult to maintain and is therefore unfeasible (Further detailed in our model section). With our model’s results, we explored alternative ideas on a biocontainment mechanism that would allow both avoiding sporulation and degrading the recombinant sequences of B. subtilis.
Spores are a morphological state that give resistance to many survival-threatening conditions, such as nutritional limitation, temperature, radiation, toxic compounds, high pressures, and competition with other microorganisms (22; 23). Evidently, cells in this state are more difficult to remove or kill. The risk of our modified spores unintentional release directly repercutes on the biosafety aspects of our foam.
Last year, the iGEM FCB-UANL 2020 team (1) worked on a system to delay sporulation through the utilization of Spo0E phosphatase, dephosphorylating Spo0A-P which at high concentrations activates sporulation. However, this year we designed a model to force the cell to enter a competence state instead of sporulation. Unfortunately, our model predicted that such a mechanism is difficult to maintain and is therefore unfeasible (Further detailed in our model section). With our model’s results, we explored alternative ideas on a biocontainment mechanism that would allow both avoiding sporulation and degrading the recombinant sequences of B. subtilis.
KILL SWITCH DESIGN
After going through the mentioned process, the idea of designing a
Kill Switch System was born (for more detail regarding the decision
making process, please go to our safety section).
Our system uses the mechanism CRISPR/Cas. Among its many advantages,
we can mention its precision and accuracy. It consists mainly of two
parts; the first one focuses on the constitutive production of RNA
guides (gRNA), while the second one is activated during a specific
moment of sporulation, killing, and avoiding spore formation without
compromising the production yield.
More information about the process of finding potential genes for its usage, as well as the search for useful information is explained in our partnership section, as we designed this mechanism as part of our partnership with the UNILA_LatAm team. In addition, the parts designed can be found in our parts section.
.
More information about the process of finding potential genes for its usage, as well as the search for useful information is explained in our partnership section, as we designed this mechanism as part of our partnership with the UNILA_LatAm team. In addition, the parts designed can be found in our parts section.
.
Design of the genetic circuit for gRNA production
Constitutive production of the gRNA is carried out through the known
strong constitutive promoter of B. subtilis P43 (BBaK143013 ).
To produce these guides, we produce them individually with the
mentioned promoter and a terminator (BBa_B0010).
Their design was carried out with the bioinformatics tool ChopChop,
which provides the different target sites, their ranking and score
according to the organism, the sequence, the Cas enzyme planned to
be used, and the cutting target (24).
The target sequences of this mechanism are separated into: sequences corresponding to a survival gene of the bacteria and recombinant DNA sequences. A total of 8 target sequences were designed in order to remove the following recombinant genes. Notice that the efficiency given by ChopChop tool is expressed as a percentage (since no sequence showed 100% efficiency, we designed more than one for the same gene).
To ensure the death of the bacteria prior to complete spore formation, three guides with the highest efficiencies (rncS-T1 72% (BBa_K4065008) , rncS-T2 71% (BBa_K4065009) were designed. Their common target was the essential gene rncS, endoribonuclease RNase III of B. subtilis, whose function is the processing of ribosomal and small RNAs from the cytoplasm, and the degradation of mRNAs of the two toxins txpA and yonT. We decided to use this target because previous experiments have shown that, if not produced correctly, 90-95% of the bacterial population will end in cell death (25; 24).
Finally, we added the strong terminator rrnb T1 designed by Randy Rettberg in 2003 (BBa_B0010).The complete circuit is shown in the next image:
The target sequences of this mechanism are separated into: sequences corresponding to a survival gene of the bacteria and recombinant DNA sequences. A total of 8 target sequences were designed in order to remove the following recombinant genes. Notice that the efficiency given by ChopChop tool is expressed as a percentage (since no sequence showed 100% efficiency, we designed more than one for the same gene).
- XylR: Regulatory protein of the Xyl promoter, activator of recombinant production of PhrC, sfp, SinI, and AbbA elements (XylR-T1 75% (BBa_K4065010), XylR-T2 70% (BBa_K4065011)).
- Cat: Protein responsible for antibiotic resistance (Cat-T1 71% (BBa_K4065014), Cat-T2 69% (BBa_K4065015)).
- Spo0F: Final part of the circuit, in order to fragment it (Spo0F-T1 65% (BBa_K4065012) Spo0F-T2 62% (BBa_K4065013).
To ensure the death of the bacteria prior to complete spore formation, three guides with the highest efficiencies (rncS-T1 72% (BBa_K4065008) , rncS-T2 71% (BBa_K4065009) were designed. Their common target was the essential gene rncS, endoribonuclease RNase III of B. subtilis, whose function is the processing of ribosomal and small RNAs from the cytoplasm, and the degradation of mRNAs of the two toxins txpA and yonT. We decided to use this target because previous experiments have shown that, if not produced correctly, 90-95% of the bacterial population will end in cell death (25; 24).
Finally, we added the strong terminator rrnb T1 designed by Randy Rettberg in 2003 (BBa_B0010).The complete circuit is shown in the next image:
Design of the genetic circuit for endonuclease and reporter protein
production
As mentioned above, the main objective of this mechanism is to avoid
the formation of recombinant spores; therefore, it is planned to
eliminate both the recombinant DNA and the mother bacteria, assuring
they won’t undergo this morphological change and our final foam will
not have remnants of genetically modified DNA.
.Sporulation is a complex process since it includes the chronological activation of approximately 500 genes in a period of approximately 7 to 8 hours, in which the spore undergoes conformational changes in 7 stages (26; 27; 28). Hence, the genetic and metabolic regulation of the sporulation process had to be deeply investigated to identify sporulation-specific promoters with high activity at the early stages of this process.
since they regulate other processes such as cannibalism, and the production of the biofilm and polysaccharide matrix (29; 30). On the contrary, the regulatory promoter pSpoIIA was chosen because it is specific to sporulation and sensitive to the RNA polymerase Sigma-H factor, which is present in the early onset stage of sporulation. The promoter (BBa_K4065000) naturally activates the spoIIA gene which is found in the second early-stage of sporulation, being responsible for triggering the irreversible process of sporulation (31; 32).
Next, the RBSs (BBa_K4065001, BBa_K4065003 , BBa_K4065004) were designed to ensure that they had the maximum possible target translation initiation rate, determined with the RBS calculator tool developed by Sallis (33).
To understand more about the regulation of sporulation and characterize the concentration of this RNA polymerase Sigma-H factor, we added a CFP reporter protein (BBa_K4065002) with an emission wavelength of 476 nm and excitation wavelength of 439 nm (34). Afterwards, the CFP protein sequence was optimized through the program GeneScript GeneSmart Codon Optimization Tool (35).
CRISPR/Cas9 enzyme (BBa_K4065005) was used due to its efficiency, capacity and accuracy with the objective of cutting the 1) rncS survival gene and 2) recombinant DNA regions. Also, biocontainment and cell death devices have achieved results in factors of 108 degradation of plasmid DNA and viable cells using this technology (36).
To ensure both cell death and plasmid and genome degradation, we included yokF (BBa_K4065007), an endonuclease derived from the prophage SPBeta, which catalyzes the hydrolysis of supercoiled DNA and RNA. It also participates in chromosomal DNA degradation and cell death by heat stress (37). The circuit can be seen in the next image.
.Sporulation is a complex process since it includes the chronological activation of approximately 500 genes in a period of approximately 7 to 8 hours, in which the spore undergoes conformational changes in 7 stages (26; 27; 28). Hence, the genetic and metabolic regulation of the sporulation process had to be deeply investigated to identify sporulation-specific promoters with high activity at the early stages of this process.
since they regulate other processes such as cannibalism, and the production of the biofilm and polysaccharide matrix (29; 30). On the contrary, the regulatory promoter pSpoIIA was chosen because it is specific to sporulation and sensitive to the RNA polymerase Sigma-H factor, which is present in the early onset stage of sporulation. The promoter (BBa_K4065000) naturally activates the spoIIA gene which is found in the second early-stage of sporulation, being responsible for triggering the irreversible process of sporulation (31; 32).
Next, the RBSs (BBa_K4065001, BBa_K4065003 , BBa_K4065004) were designed to ensure that they had the maximum possible target translation initiation rate, determined with the RBS calculator tool developed by Sallis (33).
To understand more about the regulation of sporulation and characterize the concentration of this RNA polymerase Sigma-H factor, we added a CFP reporter protein (BBa_K4065002) with an emission wavelength of 476 nm and excitation wavelength of 439 nm (34). Afterwards, the CFP protein sequence was optimized through the program GeneScript GeneSmart Codon Optimization Tool (35).
CRISPR/Cas9 enzyme (BBa_K4065005) was used due to its efficiency, capacity and accuracy with the objective of cutting the 1) rncS survival gene and 2) recombinant DNA regions. Also, biocontainment and cell death devices have achieved results in factors of 108 degradation of plasmid DNA and viable cells using this technology (36).
To ensure both cell death and plasmid and genome degradation, we included yokF (BBa_K4065007), an endonuclease derived from the prophage SPBeta, which catalyzes the hydrolysis of supercoiled DNA and RNA. It also participates in chromosomal DNA degradation and cell death by heat stress (37). The circuit can be seen in the next image.
The complete information of each part used can be found on
iGEM’s part registry, and the experimental process we followed
to the characterization of the Ranaspumin parts is available in
our results
results and engineering success sections.
REFERENCES
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fluorosurfactants. https://bit.ly/3mlaf1W
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6. Jong-Hwan, L., Myoung-Seok, K., Park Byung-Kwon, Hwang Youn-Hwan, Hwang Mi-Hyun, Park Seung-Chun, & Hyo-In, Y. (2020). Systemic Availability and Pharmacokinetics of Surfactin, a Lipopeptide Produced by Bacillus subtilis BC1212 in Rats. Toxicological Research, 21(4), 319–323.
7. Razafindralambo, H., Paquot, M., Baniel, A., Popineau, Y., Hbid, C., Jacques, P., & Thonart, P. (1996). Foaming properties of surfactin, a lipopeptide biosurfactant from Bacillus subtilis. Journal of the American Oil Chemists’ Society, 73(1), 149–151. doi:10.1007/bf02523463
8. Hayes, D., Solaiman, D., Ashby, R. (2019). Biobased Surfactantants: Synthesis, Properties, and Applications. 2nd Edition.
9. Barcelos, G. S., Dias, L. C., Fernandes, P. L., Fernandes, R., Borges, A. C., Kalks, K. H., & Tótola, M. R. (2014). Spray drying as a strategy for biosurfactant recovery, concentration and storage. SpringerPlus, 3(1), 49. doi:10.1186/2193-1801-3-49
10. Vidyasagar. A. (2016, December 22). What Are Biofilms? Livescience.com; Live Science. https://bit.ly/3vsRfCO
11. Epstein, A. K., Pokroy, B., Seminara, A., & Aizenberg, J. (2010). Bacterial biofilm shows persistent resistance to liquid wetting and gas penetration. Proceedings of the National Academy of Sciences, 108(3), 995–1000. doi:10.1073/pnas.1011033108
12. Eskuchen, R. and Nitsche, M. (1996). Technology and Production of Alkyl Polyglycosides. In Alkyl Polyglycosides (eds K. Hill, W. von Rybinski and G. Stoll). doi:10.1002/9783527614691.ch2
13. Pantelic, Ivana, and Bojana Cuckovic. "Alkyl Polyglucosides: An emerging class of sugar surfactants." Alkyl Polyglucosides. Woodhead Publishing, 2014. 1-19.
14. Schramm, Laurier L., ed. (2000). Surfactants: fundamentals and applications in the petroleum industry. Cambridge University Press.
15. Erfani, A., Khosharay, S., Flynn, N. H., Ramsey, J. D., & Aichele, C. P. (2020). Effect of zwitterionic betaine surfactant on interfacial behavior of bovine serum albumin (BSA). Journal of Molecular Liquids, 318, 114067. doi:https://doi.org/10.1016/j.molliq.2020.114067
16. Lee, C.T., Smith, K.A. & Hatton, T.A. (2005). Photocontrol of protein folding: the interaction of photosensitive surfactants with bovine serum albumin. Biochemistry, 44(2), 524-36. doi: 10.1021/bi048556c. PMID: 15641777.
17. Rani, K. (2012). Biochemical properties of bovine serum albumin. Biotech articles. https://bit.ly/3n35YR6
18. Valstar, A., Almgren, M., Brown, W., & Vasilescu, M. (2000). The Interaction of Bovine Serum Albumin with Surfactants Studied by Light Scattering. Langmuir, 16, 922-927.
19. Cooper, D.G., & Paddock, D.A. (1984). Production of a Biosurfactant from Torulopsis bombicola. Applied and Environmental Microbiology, 47(1), 173–176. doi:10.1128/aem.47.1.173-176.1984
20. Tian, Z.J., Chen, L.Y., Li, D.H., Pang, H.Y., Wu, S., Liu, J.B. & Huang, L. (2016). Characterization of a Biosurfactant-producing Strain Rhodococcus sp. HL-6. Romanian Biotechnological Letters, 21.4, 11651.
21. Eddouaouda, K., Mnif, S., Badis, A., Younes, S.B., Cherif, S., Ferhat, S., Mhiri, N., Chamkha, M., & Sayadi, S. (2011). Characterization of a novel biosurfactant produced by Staphylococcus sp. strain 1E with potential application on hydrocarbon bioremediation. Journal of Basic Microbiology, 52(4), 408–418. doi:10.1002/jobm.201100268
22. Schultz, D., Wolynes, P., Jacob, E. & Onuchic, J. (2009). Deciding fate in adverse times: Sporulation and competence in Bacillus subtilis. PNAS, 106(50). doi:10.1073/pnas.0912185106
23. Zeigler, D. & Nicholson, W. (2017). Experimental evolution of Bacillus subtilis. SFAM, 19(9). doi:10.1111/1462-2920.13831
24. Labun, K., Montague, T.G., Krause, M., Torres Cleuren, Y.N., Tjeldnes, H., & Valen, E. (2019). CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Research, 47(W1), W171–W174. doi:10.1093/nar/gkz365
25. Herskovitz, M.A., & Bechhofer, D.H. (2002). Endoribonuclease RNase III is essential in Bacillus subtilis. Molecular Microbiology, 38(5), 1027–1033.doi:10.1046/j.1365-2958.2000.02185.x
26. Eichenberger, P., Fujita, M., Jensen, S.T., Conlon, E.M., Rudner, D.Z., Wang, S.T., Ferguson, C., Haga, K., Sato, T., Liu, J.S. & Losick, R. (2004). The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis. PLoS Biol, 2(10).doi:10.1371/journal.pbio.0020328
27. Hoch, J. (1993). Regulation of the phosphorelay and the initiation of the sporulation in Bacillus subtilis. Annual Review of Microbiology, 47(1). doi:10.1146/annurev.mi.47.100193.002301
28. Tan, I. S., & Ramamurthi, K. S. (2013). Spore formation in Bacillus subtilis. Environmental Microbiology Reports, 6(3), 212–225. doi:10.1111/1758-2229.12130
29. Errington J. (2003). Regulation of endospore formation in Bacillus subtilis. Nature Reviews Microbiology, 1. doi:10.1038/nrmicro750
30. Hamon, M. & Lazazzera, B. (2001). The sporulation transcription factor Spo0A is required for biofilm development in Bacillus subtilis. Molecular Microbiology, 42(5).doi:10.1046/j.1365-2958.2001.02709.x
31. Fujita, M., Gonzalez-Pastor, J.E. & Losick, R. (2005). High and low threshold genes in the Spo0A regulon of Bacillus subtilis. Journal of Bacteriology, 187(4), 1357-68.
32. Uniprot. (2021). sigH - RNA polymerase sigma-H factor - Bacillus subtilis (strain 168) - sigH gene & protein.https://bit.ly/3lzq31U
33. Salis, H.M. (2011). The Ribosome Binding Site Calculator. Methods in Enzymology Synthetic Biology, Part B - Computer Aided Design and DNA Assembly, 19-42. doi:10.1016/b978-0-12-385120-8.00002-4
34. Guest Blogger. (2014). Choosing Your Fluorescent Proteins for Multi-Color Imaging. Addgene.org. https://bit.ly/30mGmqX
35. GenScript. (2019). GenSmart Codon Optimization. https://bit.ly/3jiDKAK
36. Caliando, B.J., & Voigt, C.A. (2015). Targeted DNA degradation using a CRISPR device stably carried in the host genome. Nature Communications, 6(1). https://doi.org/10.1038/ncomms7989
37. Uniprot. (2021). yokF - SPbeta prophage-derived endonuclease YokF precursor - Bacillus subtilis (strain 168) - yokF gene & protein. https://bit.ly/3iXynXD
2. Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2018). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 15(2), 196-204.
3. Seydlová, G., & Svobodová, J. (2008). Review of surfactin chemical properties and the potential biomedical applications. Open Medicine, 3(2). 4. Cooper, A., & Kennedy, M. W. (2017). Biofoams and natural protein surfactants. Biophysical Chemistry, 151(3), 96–104. https://doi.org/10.1016/j.bpc.2010.06.006
5. Hyo-Jick, C., & Montemagno, C. (2013). Recent Progress in Advanced Nanobiological Materials for Energy and Environmental Applications. Materials, 6(12). doi:10.3390/ma6125821
6. Jong-Hwan, L., Myoung-Seok, K., Park Byung-Kwon, Hwang Youn-Hwan, Hwang Mi-Hyun, Park Seung-Chun, & Hyo-In, Y. (2020). Systemic Availability and Pharmacokinetics of Surfactin, a Lipopeptide Produced by Bacillus subtilis BC1212 in Rats. Toxicological Research, 21(4), 319–323.
7. Razafindralambo, H., Paquot, M., Baniel, A., Popineau, Y., Hbid, C., Jacques, P., & Thonart, P. (1996). Foaming properties of surfactin, a lipopeptide biosurfactant from Bacillus subtilis. Journal of the American Oil Chemists’ Society, 73(1), 149–151. doi:10.1007/bf02523463
8. Hayes, D., Solaiman, D., Ashby, R. (2019). Biobased Surfactantants: Synthesis, Properties, and Applications. 2nd Edition.
9. Barcelos, G. S., Dias, L. C., Fernandes, P. L., Fernandes, R., Borges, A. C., Kalks, K. H., & Tótola, M. R. (2014). Spray drying as a strategy for biosurfactant recovery, concentration and storage. SpringerPlus, 3(1), 49. doi:10.1186/2193-1801-3-49
10. Vidyasagar. A. (2016, December 22). What Are Biofilms? Livescience.com; Live Science. https://bit.ly/3vsRfCO
11. Epstein, A. K., Pokroy, B., Seminara, A., & Aizenberg, J. (2010). Bacterial biofilm shows persistent resistance to liquid wetting and gas penetration. Proceedings of the National Academy of Sciences, 108(3), 995–1000. doi:10.1073/pnas.1011033108
12. Eskuchen, R. and Nitsche, M. (1996). Technology and Production of Alkyl Polyglycosides. In Alkyl Polyglycosides (eds K. Hill, W. von Rybinski and G. Stoll). doi:10.1002/9783527614691.ch2
13. Pantelic, Ivana, and Bojana Cuckovic. "Alkyl Polyglucosides: An emerging class of sugar surfactants." Alkyl Polyglucosides. Woodhead Publishing, 2014. 1-19.
14. Schramm, Laurier L., ed. (2000). Surfactants: fundamentals and applications in the petroleum industry. Cambridge University Press.
15. Erfani, A., Khosharay, S., Flynn, N. H., Ramsey, J. D., & Aichele, C. P. (2020). Effect of zwitterionic betaine surfactant on interfacial behavior of bovine serum albumin (BSA). Journal of Molecular Liquids, 318, 114067. doi:https://doi.org/10.1016/j.molliq.2020.114067
16. Lee, C.T., Smith, K.A. & Hatton, T.A. (2005). Photocontrol of protein folding: the interaction of photosensitive surfactants with bovine serum albumin. Biochemistry, 44(2), 524-36. doi: 10.1021/bi048556c. PMID: 15641777.
17. Rani, K. (2012). Biochemical properties of bovine serum albumin. Biotech articles. https://bit.ly/3n35YR6
18. Valstar, A., Almgren, M., Brown, W., & Vasilescu, M. (2000). The Interaction of Bovine Serum Albumin with Surfactants Studied by Light Scattering. Langmuir, 16, 922-927.
19. Cooper, D.G., & Paddock, D.A. (1984). Production of a Biosurfactant from Torulopsis bombicola. Applied and Environmental Microbiology, 47(1), 173–176. doi:10.1128/aem.47.1.173-176.1984
20. Tian, Z.J., Chen, L.Y., Li, D.H., Pang, H.Y., Wu, S., Liu, J.B. & Huang, L. (2016). Characterization of a Biosurfactant-producing Strain Rhodococcus sp. HL-6. Romanian Biotechnological Letters, 21.4, 11651.
21. Eddouaouda, K., Mnif, S., Badis, A., Younes, S.B., Cherif, S., Ferhat, S., Mhiri, N., Chamkha, M., & Sayadi, S. (2011). Characterization of a novel biosurfactant produced by Staphylococcus sp. strain 1E with potential application on hydrocarbon bioremediation. Journal of Basic Microbiology, 52(4), 408–418. doi:10.1002/jobm.201100268
22. Schultz, D., Wolynes, P., Jacob, E. & Onuchic, J. (2009). Deciding fate in adverse times: Sporulation and competence in Bacillus subtilis. PNAS, 106(50). doi:10.1073/pnas.0912185106
23. Zeigler, D. & Nicholson, W. (2017). Experimental evolution of Bacillus subtilis. SFAM, 19(9). doi:10.1111/1462-2920.13831
24. Labun, K., Montague, T.G., Krause, M., Torres Cleuren, Y.N., Tjeldnes, H., & Valen, E. (2019). CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Research, 47(W1), W171–W174. doi:10.1093/nar/gkz365
25. Herskovitz, M.A., & Bechhofer, D.H. (2002). Endoribonuclease RNase III is essential in Bacillus subtilis. Molecular Microbiology, 38(5), 1027–1033.doi:10.1046/j.1365-2958.2000.02185.x
26. Eichenberger, P., Fujita, M., Jensen, S.T., Conlon, E.M., Rudner, D.Z., Wang, S.T., Ferguson, C., Haga, K., Sato, T., Liu, J.S. & Losick, R. (2004). The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis. PLoS Biol, 2(10).doi:10.1371/journal.pbio.0020328
27. Hoch, J. (1993). Regulation of the phosphorelay and the initiation of the sporulation in Bacillus subtilis. Annual Review of Microbiology, 47(1). doi:10.1146/annurev.mi.47.100193.002301
28. Tan, I. S., & Ramamurthi, K. S. (2013). Spore formation in Bacillus subtilis. Environmental Microbiology Reports, 6(3), 212–225. doi:10.1111/1758-2229.12130
29. Errington J. (2003). Regulation of endospore formation in Bacillus subtilis. Nature Reviews Microbiology, 1. doi:10.1038/nrmicro750
30. Hamon, M. & Lazazzera, B. (2001). The sporulation transcription factor Spo0A is required for biofilm development in Bacillus subtilis. Molecular Microbiology, 42(5).doi:10.1046/j.1365-2958.2001.02709.x
31. Fujita, M., Gonzalez-Pastor, J.E. & Losick, R. (2005). High and low threshold genes in the Spo0A regulon of Bacillus subtilis. Journal of Bacteriology, 187(4), 1357-68.
32. Uniprot. (2021). sigH - RNA polymerase sigma-H factor - Bacillus subtilis (strain 168) - sigH gene & protein.https://bit.ly/3lzq31U
33. Salis, H.M. (2011). The Ribosome Binding Site Calculator. Methods in Enzymology Synthetic Biology, Part B - Computer Aided Design and DNA Assembly, 19-42. doi:10.1016/b978-0-12-385120-8.00002-4
34. Guest Blogger. (2014). Choosing Your Fluorescent Proteins for Multi-Color Imaging. Addgene.org. https://bit.ly/30mGmqX
35. GenScript. (2019). GenSmart Codon Optimization. https://bit.ly/3jiDKAK
36. Caliando, B.J., & Voigt, C.A. (2015). Targeted DNA degradation using a CRISPR device stably carried in the host genome. Nature Communications, 6(1). https://doi.org/10.1038/ncomms7989
37. Uniprot. (2021). yokF - SPbeta prophage-derived endonuclease YokF precursor - Bacillus subtilis (strain 168) - yokF gene & protein. https://bit.ly/3iXynXD
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