Overview
We aim to target invertase, the enzyme present in the sugarcane stem which is responsible for the degradation of sucrose into glucose and fructose. The project has been structured into the following major modules:
- A fructose-regulated anti-invertase construct
- An atmospherically-regulated kill switch
- A novel polymer inoculant
Each aspect of the project design has been explained in the proceeding sections. Click here to know more about the action of invertases.
Note: In lieu of the global COVID-19 pandemic, our institute suspended instruction and lab access from the second week of March 2020. We had begun our initial growth curve experiments prior to this but due to closure of the institute, we couldn't complete our initial plans. We hope to conduct the experiments during Phase 2.
pFruB - Cra Construct
The primary product of the action of the invertase enzyme is fructose and glucose. Hence the concentration of fructose inside the sugarcane stem is proportional to the activity of invertase and consequently the amount of invertase present. We employed this dynamic concentration of fructose to modulate the expression of anti-invertase.
We designed a genetic construct based on a biosensor created by iGEM Evry Paris-Saclay (BBa_K2448032). A biosensor is a module that combines biological molecules as the recognition element with a physical transducer or repressor and outputs quantitative data corresponding to the biomolecule’s concentration Park, M et al., 2013. Generally, these specific interactions between the ligand and the transducer are used to determine secretion or production rates in the form of an electrochemical and/or optical signals. In our case, we coupled the biosensor to the expression of anti-invertase.
pFruB - Cra (FruR) Regulation
The enterobacterial catabolite repressor/activator (Cra) protein or the FruR protein is a pleiotropic regulator that controls expression of a large number of metabolic genes in response to the flux of glycolytic intermediates in various natural biological systems. The regulator is able to interact with specifically fructose-1-phosphate and regulate the action of pFruB promoter (which is a common promoter usually found in the fructose operon) Pereira, L.F.M. et al., 2016 Chavarría, M et al., 2014.
Mechanism
The FruR expression is under an IPTG-induced pLac promoter (BBa_K3156000) which is induced before packaging the bacteria in our polymer inoculant. FruR is a transcription factor with an affinity for fructose-1-phosphate which is produced from the D-fructose formed after invertase action. The FruR gene (BBa_K2448009) encoding for the FruR protein prevents the transcription of the pFruB-regulated promoters. pFruB (BBa_K2448017) is the promoter region following FruR and is repressed by the FruR transcription factor, in the absence of D-Fructose. This prevents any further transcription, and no anti-invertase protein is produced.
Figure 1: The behaviour of the FruR-Cra construct in the absence of fructose
If D-Fructose is present in the cell, the FruR transcription factor will bind preferentially to it and thus be inactivated. This means that the repression of the related promoter pFruB will be released, enabling the transcription of the anti-invertase protein which can then act on invertase and prevent the inversion of sucrose.
Figure 2: The behaviour of the FruR-Cra construct in the presence of fructose
His Tag
The His tag is by far the most popular affinity tag for purification of recombinant proteins. Typically, the tag is composed of 6–10 consecutive histidines at either terminus of the protein of interest. These tags can be used for protein purification with the help of Nickel or Cobalt (adjacent period 4 transition metals) in sepharose/agarose based resin columns Burgess, R. R. & Deutscher, M. P., 2009. We use a His tag (BBa_K1223006) in our construct to be able to extract anti-invertase and observe its properties as well as use the protein in different planned experiments.
For the phase-2 of the project, we would like to be able to extract the protein in the lab. The invertase protein sequence we procured had an existing signalling peptide sequence to allow the bacterial chassis to secrete the protein in the extracellular matrix. We propose characterisation of this protein and if inadequate, include another strong signalling peptide in the final construct.
Atmosphere-Regulated Kill Switch
Advances in synthetic biology raise increasingly essential questions about biocontamination and biological containment. It becomes imperative that genetically-engineered organisms are not deleterious to the ecosystem and that scientists have adequate control over their production and multiplication. Certain safeguards are developed to ensure all possible safety — which are then introduced into the genetically modified organism — that compels the organism to rely on a “survival” factor to maintain viability in the population. Kill switches when introduced into the genetic material results in cellular death under certain conditions, such as the activation of a “trigger” caused by environmental changes, thus helping us effectively control the proliferation of the genetically-engineered microorganism.
We intend to introduce our strain of bacteria into the sugarcane matrix to synthesise anti-invertase. Considering the close proximity of the system with both agricultural environments and human consumables, it is indispensable that our bacteria include a lethality mechanism. We propose to use a modified type II ccdA-ccdB toxin-antitoxin system as our kill switch that is activated upon exposure to atmospheric concentrations of oxygen. A change in the oxygen concentration in the environment results in a sharp increase in the amount of the toxin protein being synthesised which disturbs the already established equilibrium between the toxin and antitoxin concentrations, effectively killing the cell, and thus preventing any contamination by stray microbes.
Type II Toxin-Antitoxin Systems
Bacterial toxin-antitoxin systems were characterised in the 1980s as molecular systems encoded in plasmids that ensured the persistence of a plasmid in the host lineage. The only daughter cells that survive are the ones that contain the plasmid. This phenomenon is also known as post-segregational killing. Most prokaryotic chromosomes contain a number of toxin-antitoxin modules that consist of a pair of genes that encode for two components: a stable toxin protein and its unstable, labile cognate antitoxin. Toxins are always proteins, but the antitoxins can either be RNAs (in type I and III systems) or proteins (in type II systems) Syed, M. & Lévesque, C., 2012.
Type II toxin-antitoxin systems use a protein antitoxin to deactivate the toxin protein via protein-protein interactions. All type II antitoxins are dual-function, two-domain proteins that consist of a protein-protein interaction domain and a DNA-binding domain. The antitoxin binds to the toxin and inhibits its activity, and the stable TA complex binds to the operator of the corresponding TAS~Toxin-Antitoxin System operon via the DNA-binding domain of the antitoxin and represses its transcription Makarova et al., 2009 Tam & Kline, 1989.
As our product would be introduced into a plant which is later converted into a consumable item, we decided to use the ccdA-ccdB toxin-antitoxin system as the kill switch for its relative non-toxicity towards humans and more generally, mammalian cells Reschner et al., 2013.
ccdA-ccdB System
In the ccdA-ccdB system, the two genes are organised in an operon in which the antitoxin ccdA gene is loacted upstream of the toxin ccdB gene. ccdA prevents ccdB toxicity by forming a tight ccdA-ccdB complex Afif et al., 2001. ccdB inhibits DNA replication by poisoning DNA gyrase; more specifically, ccdB prevents DNA gyrase from disentangling DNA from replication resulting in double-stranded breaks in the DNA Dao-Thi et al., 2005. ccdA and ccdB are able to form complexes of various stoichiometries depending on their molar ratios.
Our kill switch is a modified version of the ccdA-ccdB TA system (BBa_K3512002, BBa_K3512001), where the ccdB (toxin) CDS is placed under a T7 (BBa_I719005) constitutive promoter, and the ccdA (antitoxin) CDS is placed under an FNR promoter (BBa_K1123000) whose activity is controlled by the concentration of oxygen present in the environment.
FNR Promoter
This part we used is a standard FNR promoter. The promoter is used in E. coli strains to induce protein expression in anaerobic environments. It is natively used to induce metabolic processes when the E. coli enter anaerobic environments. The part is often used to express any protein sequence placed behind it in such environmental conditions. On entering a hypoxic environment, the formation of \( [ 4Fe-4S]^{2+} \) dimers is stimulated within the FNR-producing bacteria. These dimers are the transcription activators of the FNR promoter. They are able to bind to the promoter, thus allowing it to become active.
As the environment inside sugarcane is hypoxic compared to the external atmosphere, we chose to place the ccdA antitoxin under the FNR promoter. The mechanism of the kill switch has been explained in the next section.
Mechanism
The kill switch function is best explained with the help of two different cases: the first where the bacteria is present in a sufficiently oxygenated environment, and the second where it is present in a hypoxic one.
Hypoxic Environment
The ccdB toxin is under a constitutive promoter which constantly expresses the toxin inside the cell. Due to prevalent hypoxic conditions inside the sugarcane Bieleski, R. L., 1960 conditions, the FNR promoter will be active and hence the ccdA antitoxin will also be produced in the cell. Since both ccdA and ccdB are present in the cell, they form a highly stable toxin-antitoxin complex. This way the bacteria is able to survive inside the sugarcane stem.
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Figure 3: Kill switch construct in a hypoxic environment
Aerobic Environment
In this case, too, ccdB is expressed constitutively with the help of the T7 promoter; however, due to the presence of oxygen, the FNR promoter is switched off. This leads to an increase in the levels of ccdB in the cell which in turn leads to a complete halt in cell division and subsequently its death. This ensures that the bacteria will not be able to survive outside the sugarcane environment.
Figure 4: Kill switch construct in an aerobic environment
Plasmid Design
We plan to use the pET28a(+) plasmid for our application.
Figure 5: Plasmid for the pFruB-Cra Module
Figure 6: Plasmid for the Atomsphere-regulated Kill Switch
Polymer Inoculant
We have devised a novel polymer inoculant to create an easy-to-use, robust and organic delivery mechanism of our bacteria. The inoculant consists of a Carboxymethylcellulose based polymer, bentonite suspending agent and sorbic acid preservative. We have also optimized the viscosity to minimize the innoculant’s toxicity, and ensured that it is close to that of water for efficient transport. This inoculant would be supplied to the farmer and can be used through an injector mechanism. In this manner the sugarcane’s inherent transport mechanism due to transpirational pull is leveraged for the uniform distribution of the bacteria through the plant.
For the large scale manufacturing of this inoculant we have constructed a process flow diagram to emulate an industrial setting. The assembly consists of an anaerobic countinuous stirred tank bioreactor, a mixing tank, a cross-flow filter unit and a sterilization unit. Through diffusivity and mass-flux calculations, we hope to able to compute the concentration of bacterial cells needed in the inoculant to ensure effective inactivity of invertase as a part of phase 2.