Team:IISER-Tirupati India/Proof Of Concept
OviCloak is aimed to be a non-hormonal and environment-friendly approach to Long-Acting Reversible Contraception (LARC). OviCloak achieves this through the heterologous production of recombinant proteins and acts as an effective and reversible method of contraception.
We tested our hypothesis rigorously through a combination of mathematical modelling, protein modelling and wet lab experiments. We used mathematical modelling to develop models for delivery and colonisation of the bacteria and performed wet-lab experiments to test the novel gene cassettes for each of its attributes. Due to limited accessibility to the laboratory owing to pandemic restrictions we used modelling studies to our advantage to test the feasibility of our project.
PRINCIPLE
OviCloak achieves contraception by mimicking a naturally occurring process during fertilisation that prevents polyspermy by making the ovum impermeable to sperm. The bacteria are genetically engineered to produce ovastacin, a protease molecule that specifically cleaves the ZP2 protein of the Zona Pellucida layer surrounding the ovum. Ovastacin is a naturally produced protein by the ovum and is released from the cortical granules of the ovum upon penetration by the sperm to prevent polyspermic fertilisation. We designed OviCloak to mimic this natural reaction and induce hardening of the ovum prior to fertilisation.
To minimise the possibilities of any side effects due to the introduction of genetically modified bacteria, we chose Lactobacillus acidophilus, a commensal residing in the upper reproductive tract for heterologous production of ovastacin. However, for providing the proof of concept, we used Bacillus subtilis, as our model organism, another gram-positive bacterium, as they are well-characterized and genetically amenable.
To learn more about the choice of commensal, see Safety.
To ensure reversibility of the contraceptive for providing the end-users control over their fertility we designed an inducible Kill switch to reverse the effects of contraception. The Kill switch can be induced through the administration of an inducer which can be delivered using the same technique used to introduce the bacteria according to the will of the user and environmental conditions.
GOALS
Module-1: Delivery and Colonisation
- Delivery of GM bacteria to the ampullary region to colonize on the walls of the fallopian tube from the inside.
Module-2: Protease Production
- Ovastacin regulation by SRTF1-P22-Ovastacin genetic circuit: To regulate ovastacin, a Zinc metalloprotease, production with help of the hormonal changes in the menstrual cycle.
- The cleavage assay: To show that ovastacin produced by the genetically modified bacteria is capable of cleaving human ZP2 through an in-vitro cleavage assay experiment.
Module-3: Kill switch
- Kill switch for reversibility: To demonstrate the working of an external inducer-based Kill switch when the user wishes to become fertile again.
- Kill switch for biosafety: To Kill the bacteria when it gets expelled out of the reproductive system to the external environment.
MODULE 1: DELIVERY AND COLONISATION
Since the ampullary region in the oviduct serves as the site of fertilisation, the bacteria should be delivered to this region. This delivery of the bacteria to the ampulla can be achieved with the use of a catheter which assists in the delivery of the bacteria to the specified site. This procedure is minimally invasive. The catheter is not a unique tool, this is used in hospitals for other medical procedures as well. Since the Lactobacillus acidophilus is immotile, it would adhere to the mucus layer once released and selectively colonise the region.
The number of bacteria needed to be delivered to achieve effective contraception is theoretically determined and is dependent on the following factors:
The fallopian tube has a carrying capacity that determines the maximum load of microbes that the region can take. After initial delivery, the bacterial population attains saturation post-colonisation, which is the final population.
Lactobacillus secretes lactic Acid, hydrogen peroxide, and Bacteriocins which suppresses the growth of other bacteria which would disturb the microflora in that region.
The ovastacin level near the ovum is dependent upon the distance of the ovum from the site of production, half-life, diffusion, and reactivity of ovastacin in the fallopian tube environment.
Considering these factors the delivery of bacteria is optimised to produce the required amount of ovastacin to prevent fertilisation. The colonisation of the bacteria is explained using the continuous Haldane logistic equation which predicts the growth kinetics closer to real-world growth curves.
Thus the final bacterial population produces ovastacin and reaches the ovum in synchrony within the ovulation window at an optimum level for the zona hardening.
For more details, see Model
MODULE 2: PROTEASE PRODUCTION
Overview
The idea behind OviCloak is heterologous protein production by genetically modifying commensal bacteria residing in the fallopian tube. The protein being produced in our case is ovastacin, a zinc metalloprotease. Ovastacin recognises a specific sequence of amino acids of the ZP2 protein and cleaves it. This cleavage leads to zona hardening which prevents fertilisation, such that sperms are not able to fertilise the ovum.
Bacillus subtilis has been chosen as a gram-positive model organism to secrete recombinant human ovastacin. It also has a Tat secretory pathway which can secrete folded proteins into the extracellular environment. [1]
To give proof of this concept in our laboratory, we wanted to replicate this scenario in-vitro with the help of heterologous protein production and purification from bacterial and yeast clones. To achieve this, we came up with a holistic experimental design that would be carried out in the laboratory to give concrete proof that ovastacin secreted from Bacillus subtilis would cleave the ZP2 protein at its respective site and cause the subsequent zona hardening.
Moreover, as opposed to a constitutive production, we wanted to regulate the production of ovastacin. This was done to stop the unnecessary production of ovastacin, which can potentially harm the surrounding environment.
During the menstrual cycle, there is a shift in the concentration of progesterone in the tubular fluid from the ovulatory phase to the luteal phase. (see Background). We chose this shift in concentrations of progesterone to repress the unnecessary production of ovastacin. Thus, with our experimental design, we wanted to show the repression of ovastacin production due to the rise of progesterone levels in the surroundings.
Experimental design
The experimental design centers around molecular cloning and protein expression and purification. The heterologous protein production would be achieved in Bacillus subtilis as the model organism and yeast as the support organism.
Hormone regulation
Our proof of concept for protease production is to show that the ovastacin protease is produced under hormonal regulation. This is based on the working and interaction of the SRTF1 transcription factor and the P22 repressor protein molecule.
The SRTF1 is a bacterial transcription factor that has been shown to respond to rising levels of progesterone in a concentration-dependent manner [2]. The SRTF1 transcription factor would be present in the system to respond to the rise in the level of progesterone and the P22 repressor would subsequently repress the production of ovastacin.
To show this experimentally, we designed our genetic cassettes with different reporters such that we can check the expression and interaction of these three cassettes in a time-efficient way:-
- The SRTF1 transcription factor is linked with the mCherry reporter. Regardless of the progesterone concentration, the fluorescence produced by mCherry would be constant.
- The P22 repressor is upstream to the Azurite reporter and would produce blue fluorescence when expressed in the presence of progesterone. The SRTF1 would bind to progesterone, and thus, P22 protein is expressed. This would lead to an increase in the blue fluorescence level.
- When the progesterone concentration is increased, causing increased expression of the P22 protein, the ovastacin gene would be repressed. The ovastacin gene is upstream of the sfGFP gene, which produces green fluorescence. Thus, in higher progesterone levels, a decrease in the green fluorescence level would be observed.
We were able to test the above cassette in wetlab. See Results to know more.
We can check the relative fluorescence levels (Green(sfGFP), Red(mCherry), Blue(Azurite)) under different test conditions to determine the efficiency of interactions, if any.
For more details, see Blueprint.
These different reporters would help us identify the interactions between the different cassettes in B. subtilis. Thus, we could effectively test the production of ovastacin under hormonal regulation.
This would allow us to predict how the bacteria would produce ovastacin under the different phases of the menstrual cycle.
For the production of functional ovastacin in the bacterial system, we made use of phosphomimetics at the site of phosphorylated residues.
Model
Due to restricted lab access and insufficient time, we were not able to test this system of hormonal regulation in B. Subtilis. However, through mathematical modelling, we were able to predict the working of the genetic circuit that has been proposed.
Graphs
Cleavage assay
To prove that the bacterially derived ovastacin is able to cleave the human ZP2 protein (ZP2), we planned a cleavage assay experiment.
The cleavage assay is designed to give in-vitro proof of concept for the Zona-hardening reaction :-
- We obtained the clones of human ZP2 protein in E.Coli from Prof Satish K Gupta, National Institute Institute of Immunology, India and Dr Gagandeep Gahlay from Guru Nanak Dev University, India. Our plan is to express and purify the recombinant ZP2 protein from the said clones. The proteins would be purified in their native state through IMAC and would be without Post Translational Modifications.
- The purified ZP2 protein would be incubated with the bacterially produced ovastacin. As the ovastacin recognises a specific sequence of the ZP2 protein, it would be able to cleave the ZP2 protein at it’s specific site and produce two smaller fragments of about 90 kDa and 30 kDa.
- The cleaved ZP2 fragments would be visualised by performing immunoblot against the uncleaved ZP2 as a control.
This cleavage assay would give us definite proof that bacterially produced ovastacin is functional in-vitro.
If the aforementioned cleavage assay doesn’t show the desired results, we have come up with supplementary experiments involving Saccharomyces cerevisiae.
The genetic cassette of Saccharomyces cerevisiae has been designed to produce both ZP2 and ovastacin under different inducers. The expression of ZP2 and ovastacin are under the inducible expression of Copper (Cu2+) ions and galactose respectively.
These proteins can be produced in Saccharomyces cerevisiae to show in-vivo cleavage of the ZP2 protein and help in troubleshooting:-
- The ZP2 protein will be expressed in Saccharomyces cerevisiae and detected with the help of immunoblot and anti-FLAG antibody. The purified protein will be incubated with bacterially produced ovastacin and the cleavage assay will be performed. If the desired results are shown, we could hypothesize that ovastacin requires active ZP2 (with PTMs) to perform its function.
- If the above-described experiment doesn’t show the desired results, then we will move on to perform the
The in-vivo cleavage in Saccharomyces cerevisiae should also give smaller fragments of the ZP2 protein which can be visualized against the uncleaved ZP2 control if the cleavage is successful. This will demonstrate that ovastacin is active in in-vivo conditions.
MODULE 3: KILL SWITCH
Overview
KILL SWITCH 1
One of the most important attributes of contraceptives is the reversal at will, which fulfils the user's right to bodily autonomy. We plan to achieve this feat by our holistic experimental design and mathematical models supporting it. The Kill switch for reversibility is induced through Xylose, a monosaccharide molecule that is generally not found in the fallopian tube environment. On the wish of the user, the Xylose molecules would be introduced in the fallopian tube environment through hysteroscopic techniques, which would Kill the genetically modified bacteria in the fallopian tube and reverse the contraception.
KILL SWITCH 2
Another important aspect for OviCloak has been environmental safety as it is aimed to be an eco-friendly method of contraception. We wanted to make sure that the genetically modified bacteria would not be able to survive in the outside environment for long if there’s an accidental release. For this reason, we came up with another Kill switch which is visible light-inducible.
Experimental design for Kill Switch 1
The cassettes for the Kill switch have been designed to check the induction of toxin upon the introduction of Xylose in the surroundings.
The gene cassettes consist of XylR repressor, which restricts the production of YqcG toxin. When Xylose enters the surroundings of the bacteria, the XylR repressor interacts with Xylose, and thus, induces the production of the toxin.
In addition to this, we have also designed a cassette for YqcF antitoxin production to prevent any leaky expression of the toxin from accidentally killing the bacteria. The YqcF and YqcG proteins interact to form a toxin-antitoxin complex in the absence of Xylose.
When there is an increase in the Xylose concentrations, the increased production of the YqcG toxin leads to the death of bacteria.
To test this system, we would replace the YqcG gene with sfGFP and then check the fluorescence of the system in presence and absence of Xylose. The fluorescence intensity when the system is under uninduced conditions helps in optimizing YqcF to nullify the leaky expression of YqcG. This method has been chosen as we cannot check the working of the promoter with the toxin-producing gene without killing the bacteria.
Model for Kill Switch 1
Through our mathematical model, we were able to test the system in-silico.
Experimental design for Kill Switch 2
This Kill switch has been specially designed to trigger bacterial death in case of accidental release of bacteria in the environment. This Kill Switch will be visible light (450-500 nm) inducible.
The gene cassettes have been made with bpDNase1 which is the toxin that causes DNA degradation. Its leaky expression is negated by the antitoxin mf-Lon protein. The mf-Lon protein is able to form a complex with the toxin bpDNase1 and inactivate it.
The expression of YtvA is under a constitutive promoter. The activity of the promoter can be determined by checking mCherry reporter fluorescence which is linked to YtvA. The bpDNase is kept downstream of the pgsiB promoter. pgsiB is activated by the sigB transcription factor.
In the absence of blue light, the leaky activity of pgsiB can be checked by having a reporter gene like sfGFP downstream to it and checking its fluorescence. On exposure to blue light, the activity of pgsiB can be determined in a similar way.
Finally, by integrating all the aforementioned cassettes in a circuit, quantification of the efficiency of the Kill switch can be determined.
The next step would be to check the working of the bpDNase1- mf-Lon interaction. Through our partnership with Team IOANNINA, we have been able to come up with a set of experiments to show the working of this system in a model organism.
See Partnerships
Model for Kill Switch 2
Getting the desired results in the above-said experiments will give a definite proof of concept of a bacterial system that is able to do heterologous production of recombinant ovastacin under hormonal regulation of the menstrual cycle.
Moreover, the working kill switches will also indicate that this method of contraception is considerably reversible and doesn’t pose a significant threat to the environment.
Overall Proof Of Concept of OviCloak
REFERENCES
- Kolkman, M. A., van der Ploeg, R., Bertels, M., van Dijk, M., van der Laan, J., van Dijl, J. M., & Ferrari, E. (2008). The twin-arginine signal peptide of Bacillus subtilis YwbN can direct either Tat- or Sec-dependent secretion of different cargo proteins: secretion of active subtilisin via the B. subtilis Tat pathway. Applied and environmental microbiology, 74(24), 7507–7513.
- Grazon, C., Baer, R. C., Kuzmanović, U., Nguyen, T., Chen, M., Zamani, M., ... & Galagan, J. E. (2020). A progesterone biosensor derived from microbial screening. Nature communications, 11(1), 1-10
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