Team:IISER-Tirupati India/Blueprint


Ovi-Cloak

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Project OviCloak is about developing a novel method of contraception with the help of synthetic biology. Synthetic biology will allow us to replicate a naturally occurring process to harden the outer layer of the ovum that is called zona pellucida and provide an effective block to fertilization. The bacteria will produce a human protease called ovastacin which is a zinc metalloprotease. This protease specifically cleaves the ZP2 protein and initiates the zona hardening reaction. The production of ovastacin is under hormonal regulation and will change according to the phases of the menstrual cycle.

In addition, the bacteria are also engineered with Kill Switches, one for the reversibility of contraception and the other for Biosafety.

All the attributes of OviCloak require designing genetic cassettes that will bring about the desired results. So, here is a blueprint of the entire project to explain the different modules of our project through the genetic circuits associated with each of them.

Genetic Circuits

Ovastacin Production

Module 2 of our project aims to engineer Bacillus subtilis 168 to regulate the production and secretion of ovastacin by the changing levels of progesterone in the tubal fluid. During the menstrual cycle, there is a rise in progesterone concentration. This rise in progesterone levels will stop the unnecessary production of ovastacin. Thus, the current system was devised with repressors and transcription factors to get the required output.

Progesterone sensing

In order to stop the production of ovastacin in response to the rising concentration of progesterone, the bacteria should be able to respond to its rising concentrations. To achieve this, the following genetic circuit has been designed:-

  1. The Steroid Responsive Transcription Factor (SRTF1) is a bacterial transcription factor that can respond to progesterone in a concentration-dependent manner. [2,3] srtf1 has been placed under a constitutive promoter SP126 [1] along with a medium-weak Ribosome Binding Site (RBS) (BBa_K1351033). This is done to control the expression of the P22 repressor.
  2. The P22R gene that codes for the P22 repressor is under a synthetic constitutive promoter SP200 [1] and a medium RBS (BBa_K1351031). A binding site of SRTF1 was added to make it an inducible promoter, which is induced by progesterone.
  3. The gene for ovastacin (ASTL) is under a constitutive promoter P43 with a moderately strong RBS (BBa_K1351030). Additionally, there is also a P22 binding site to have the ovastacin production regulated by the P22 repressor.
  4. All the promoters, RBS, and binding sites with effective Kd [3,4] were chosen based on preliminary results to get the desired output.

Working of the genetic circuit

In low progesterone concentration or absence of progesterone :

When progesterone is not in the surrounding of the bacteria, then SRTF1 is produced under its constitutive promoter. SRTF1 will inhibit the production of P22 repressor protein by binding to its respective site and stopping the transcription of the P22 repressor. Thus, as a result, ovastacin will be produced and secreted out of the bacterial system.

In high progesterone concentration or presence of progesterone :

When progesterone is present in the system, it will bind to the SRTF1 transcription factor. Thus, SRTF1 will no longer be able to repress the production of the P22 repressor. P22 repressor will be expressed and go and bind to its respective site and stop the transcription of the ASTL gene. This will in turn stop the production of ovastacin protein in the bacterial system.

Working of the genetic circuit for ovastacin production and hormone sensing.

Ovastacin - ZP2 interaction

After its production in low progesterone concentrations, the ovastacin protein will go and interact with the ZP2 protein. The ZP2 protein is one of the glycoproteins found on the zona pellucida layer of the human ovum. Ovastacin recognizes a specific amino acid sequence of the ZP2 protein and cleaves the ZP2 protein at that site. The cleavage leaves two smaller fragments held together by disulfide bonds.

For more information, see Background.

Post Translational Modifications in ovastacin 

Ovastacin is a protease produced by humans and is present in its active form. To produce functionally active ovastacin from bacterial systems, we have come up with the use of phosphomimetics. These phosphomimetics are present at the sites of PTMs in the protein.

We have come up with two versions of the ovastacin protein. Ovastacin A (BBa_K3889022) has phosphomimetics for serine residues only. While ovastacin B (BBa_K3889023) has phosphomimetics for both serine and tyrosine residues. 

Signal Peptide

Proteins have target signals called signal peptides to translocate the protein extracellularly via their secretory pathways. Research in this area shows that the Bacillus subtilis possesses secretory pathways such as the general secretory (Sec) pathway and twin-arginine translocation (Tat) pathway. Sec pathway generally secretes unfolded proteins whereas the Tat pathway requires folded protein to secret them out.

The efficiency of a signal peptide to secrete a protein out of the cell has no correlation with the strength of the promoter which is located upstream of the signal peptide. Also, experimental studies have shown that the efficiency of the signal peptide partly depends on the target protein to be secreted out and that there is no universal signal peptide that can facilitate the high secretion rate of any protein.

Considering that functional and thus, folded ovastacin needed to be secreted to the extracellular environment for diffusing to the ovum cell, we plan to exploit the Tat signal pathways of Bacillus subtilis using one of these two signal peptides: PhoD (BBa_K3889050) and YwbN(BBa_K3889051). 

The signal peptide will allow the secretion of folded ovastacin to the outside environment. This will allow the protease to interact with the ZP2 protein of the zona pellucida. 

The reason for choosing these peptides specifically was that both of these were studied relatively extensively and shown to be functional. PhoD has been shown to be quite a strong signal peptide, while YwbN has been shown to be exclusively Tat secretory signal peptide [15,16,17].

Ovastacin secreted with YwbN signal peptide
Ovastacin secreted with PhoD signal peptide

Intein

In order to produce ovastacin during a limited window, i.e. when progesterone is absent and estrogen levels increase at the end of the follicular phase, we plan to engineer the bacteria with a self-splicing protein, estrogen-sensitive Sce VMA intein to induce the production of ovastacin upon induction by estrogen.

Inteins are protein splicing elements of the vacuolar ATPase subunit of S.cerevisiae and aim to utilize a chimeric intein with hER (human estrogen receptor) to produce ovastacin[18].

In its native state with intein, ovastacin is inactive. However, in presence of 𝛃 estradiol, the N and C terminals of the intein will self catalyze its splicing and ligate the N and C terminals of the ovastacin present on the flanking ends of the intein. This would result in the production of the active form of ovastacin. 

This additional feature to the production of ovastacin adds an additional layer of specificity and could potentially decrease the metabolic burden imposed on the GMO.

Even though we couldn’t test this design via any simulations or experiments, this part of the module stands as a promising future prospect and improvement to the project.

Estrogen-sensitive chimeric intein with hER and inactive ovastacin
Fig 1. Estrogen-sensitive chimeric intein with hER and inactive ovastacin
Working of Intein protein.

Kill Switch for reversibility

This Kill switch has been designed to kill the bacteria for the reversibility of contraception. As the reversibility of the contraception would occur on the will of the user, the kill switch is inducible. Moreover, the inducer has been chosen, such that it is generally not present in the fallopian tube environment.

Xylose is a sugar molecule that is generally not present in the fallopian tube environment and is also able to regulate the production of proteins in the bacterial cell through the XylR repressor that specifically responds to xylose. This repressor is coded by the xylR gene. Thus, we constructed a type 2 toxin-antitoxin system under a xylose inducible system[5].

The design of the genetic circuit is as follows:-

  1. The toxin-producing gene yqcG is regulated by the xylose inducible promoter PXylA.
  2. The antitoxin-producing gene yqcF is under the synthetic constitutive promoter SP126 and (BBa_K1351031). This will ensure constant production of antitoxin YqcF. This YqcF antitoxin is produced to form a complex with the toxin YqcG and thus prevents any leaky expression of the toxin from killing the bacteria.
  3. xylR is under a constitutive promoter P43 and RBS (BBa_K1351031). This will ensure constant suppression of the toxin when there is no xylose in the system. XylR is the repressor responsible for the inducibility of PXylA.
Working of the genetic circuit for xylose inducible kill switch.
DNA degradation due to Xylose uptake

Working of the genetic circuit

In absence of Xylose :

The XylR repressor molecule is produced under its constitutive promoter and represses the production of the YqcG toxin. Even then, there is evidence of leaky expression of the YqcG toxin. Thus, the constitutive expression of antitoxin YqcF will inactivate the toxin by forming a complex with it. This toxin-antitoxin system and repression of the toxin of XylR prevent the bacteria from dying in the absence of xylose.

In presence of Xylose :

When xylose is present in the system, it acts as an inducer for the production of the YqcG toxin. The xylose molecules interact with the XylR repressor and inactivate it. This will, in turn, lead to the production of the toxin YqcG. The concentration of YqcG toxin then exceeds the concentration of the anti-toxin YqcF. YqcG acts as DNase and leads to the degradation of DNA present inside the bacterial cells. The cells now contain all the other biomolecules other than DNA in functional form. For a few hours after induction of YqcG, almost all the population loses its DNA but the RNA and proteins remain unharmed. Due to this the membrane and cell division ability of the cells remain functional until the molecules degrade by themselves based on their half-lives. Such cells were named DNA-less cells (DLCs) by Elbaz et al (2015) [6]. DLCs were able to continue normal physiologies for as long as 24 hrs. Similarly, Ackermann et al (2016) overexpressed yqcG and found that a decrease in CFU, and after 48 hours only suppressor cells containing mutations in the yqcG gene were able to grow[7].

Kill Switch for Biosafety

This kill switch has been introduced to the bacterial system to ensure that the genetically modified organism doesn’t survive in the outside environment. This is done keeping in mind, the safety aspect of our project and to prevent environmental contamination due to accidental release of the bacteria. This kill switch has been made visible light-inducible and thus, the genetically modified bacteria will not be able to survive in the outside environment where visible light is readily available. Thus, we designed a novel toxin-antitoxin system which is light inducible.

To achieve this, a light-inducible toxin-producing genetic circuit has been designed with the following components:-

  1. The expression of YtvA is under P43 constitutive promoter (BBa_K143013). The ytvA gene is downstream to RBS (BBa_K1351031) and upstream to a double terminator (BBa_B0015). 
  2. The gene for producing the toxin bpDNase1 called bovine pancreatic cDNase 1 is kept downstream to pgsiB promoter. The pgsiB promoter (BBa_K3889013) is activated by the sigB transcription factor. The bpDNase1 (BBa_K3889027) produces the toxin bpDNASE 1 and upstream to double terminator (BBa_B0015).
  3. The gene mf-lon that codes for the anti-toxin mf-Lon has been kept downstream to a constitutive promoter of Bacillus subtilis SP 146 (BBa_K3889011) and upstream to a double terminator (BBa_B0015) .The mf-Lon anti-toxin forms a complex with the bpDNase1 and prevents the leaky expression of bpDNASE 1 from killing the bacteria. 
Working of the genetic circuit for visible light inducible kill switch.
Cascade reaction initiated by active YtvA
Fig 2. Cascade reaction initiated by active YtvA
DNA degradation due to light

Working of the genetic circuit

In absence of visible Light (450 nm - 500 nm)

When there is no visible light reaching the genetically modified bacteria, YtvA is not activated. Thus, the sigB factor is not activated and thus, the bpDNASE1 toxin is not produced.

Even if there is a leaky expression of bpDNASE 1, it will be inactivated by the mf-Lon anti-toxin which is present in the system, irrespective of light. 

In presence of visible light (450 nm- 500 nm)

In the presence of light, the produced YtvA is activated. The activated YtvA is in turn able to activate the sigB transcription factor. 

YtVA phosphorylates the RsbR-RsbS-RsbT complex. This releases the RsbT molecule, which goes ahead and forms a complex with the RsbU molecule. 

This RsbU-RsbT complex then dephosphorylates the RsbV molecule. Initially, SigB is inactivated by forming a complex with another molecule called RsbW. As the RsbV molecule is now dephosphorylated, it binds with RsbW. This interaction ultimately leads to the activation of the SigB transcription factor. 

The SigB transcription factor is now activated and binds to its respective site. Thus, inducing the production of the toxin bpDNASE1 under the pgsiB promoter. As the bpDNASE1 concentration exceeds that of the anti-toxin, it will degrade the DNA of the bacteria and ultimately kill the bacteria on exposure to visible light. 

Ovastacin & ZP2 in S.cerevisiae

The genetic circuit of yeast was designed to perform supplementary experiments for the proof of concept and help in troubleshooting. This genetic circuit has been devised to perform an in-vivo cleavage assay of ZP2 proteins via recombinant human ovastacin produced by yeast and/or bacteria. 

To achieve this, control over the production of respective proteins was required. To control the protein production by Saccharomyces cerevisiae, different inducible promoters were used for the induction of ZP2 and ovastacin. Thus, both the proteins can be induced separately or simultaneously according to the situation. 

The genetic circuit for yeast has been designed with the following parts :- 

  1. The ZPA gene, which is responsible for the ZP2 protein, is downstream to CUP1 which is a copper (Cu2+ ) inducible promoter[8,10]. This promoter will start producing the ZP2 protein with a Cu2+ rich media. 
  2. The Ovastacin gene(ASTL) is present downstream to the GAL1 promoter which is inducible by galactose[8,9].
  3. Both genes are equipped with same Kozak sequence i.e. BBa_J63003 [10] while Tmini (BBa_K2314608) [11] and TADH1 (BBa_J63002) [12] are the terminators of ZP2 and ovastacin respectively. 
Diagrammatic representation of Yeast genetic circuit
Fig 3. Diagrammatic representation of Yeast genetic circuit

Working of the genetic circuit

  1. The presence of Cu2+ ions in the media will induce the production of ZP2 protein in yeast. The ZP2 protein, being produced in a eukaryotic system should have all post translational modifications.
  2. In the presence of Galactose in the media, the ASTL gene will be induced, giving a functionally active ovastacin protein. This ovastacin protein being produced in a eukaryotic system, should be with Post translational modifications, properly folded and thus active. 

When both galactose and Cu2+ ions are present in the media, there will be induction of both ZP2 and ovastacin. Thus, there will be an in-vivo cleavage of the ZP2 protein by the ovastacin protease.


Diagrammatic representation of Yeast genetic circuit

References 

  1. Liu, D., Mao, Z., Guo, J., Wei, L., Ma, H., Tang, Y., Chen, T., Wang, Z., & Zhao, X. (2018). Construction, Model-Based Analysis, and Characterization of a Promoter Library for Fine-Tuned Gene Expression in Bacillus subtilis. ACS Synthetic Biology, 7(7), 1785–1797. https://doi.org/10.1021/acssynbio.8b00115 
  2. Grazon, C., Baer, R. C., Kuzmanović, U., Nguyen, T., Chen, M., Zamani, M., Chern, M., Aquino, P., Zhang, X., Lecommandoux, S., Fan, A., Cabodi, M., Klapperich, C., Grinstaff, M. W., Dennis, A. M., & Galagan, J. E. (2020). A progesterone biosensor derived from microbial screening. Nature Communications, 11(1). https://doi.org/10.1038/s41467-020-14942-5
  3. Baer, R. Cooper (2020). Discovery, characterization, and ligand specificity engineering of a novel bacterial transcription factor inducible by progesterone Boston University School of Medicine, 801 Massachusetts Avenue Suite 400 Boston, MA 02118
  4. Retrieved from: https://hdl.handle.net/2144/41109 
  5. Watkins, D., Hsiao, C., Woods, K. K., Koudelka, G. B., & Williams, L. D. (2008). P22 c2 Repressor−Operator Complex:  Mechanisms of Direct and Indirect Readout. Biochemistry, 47(8), 2325–2338. https://doi.org/10.1021/bi701826f 
  6. Brantl, S., & Müller, P. (2019). Toxin⁻Antitoxin Systems in Bacillus subtilis. Toxins, 11(5), 262. https://doi.org/10.3390/toxins11050262 
  7. Elbaz, M., & Ben-Yehuda, S. (2015). Following the Fate of Bacterial Cells Experiencing Sudden Chromosome Loss. MBio, 6(3). https://doi.org/10.1128/mbio.00092-15
  8. Bloom-Ackermann, Z., Steinberg, N., Rosenberg, G., Oppenheimer-Shaanan, Y., Pollack, D., Ely, S., Storzi, N., Levy, A., & Kolodkin-Gal, I. (2016). Toxin-Antitoxin systems eliminate defective cells and preserve symmetry in Bacillus subtilis biofilms. Environmental Microbiology, 18(12), 5032–5047. https://doi.org/10.1111/1462-2920.13471
  9. Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology, 4(9), 975–986. https://doi.org/10.1021/sb500366v
  10. http://parts.igem.org/Part:BBa_J63006 (PGAL1)
  11. http://parts.igem.org/Part:BBa_K2165004 (PCUP1)
  12. http://parts.igem.org/Part:BBa_J63003 (yeast Kozak sequence)
  13. http://parts.igem.org/Part:BBa_K2314608 (Tmini)
  14. http://parts.igem.org/Part:BBa_J63002 (TADH1)
  15. 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. https://doi.org/10.1128/AEM.01401-08
  16. Peng, C., Shi, C., Cao, X., Li, Y., Liu, F., & Lu, F. (2019). Factors Influencing Recombinant Protein Secretion Efficiency in Gram-Positive Bacteria: Signal Peptide and Beyond. Frontiers in bioengineering and biotechnology, 7, 139. https://doi.org/10.3389/fbioe.2019.00139
  17. Frain, K. M., van Dijl, J. M., & Robinson, C. (2019). The Twin-Arginine Pathway for Protein Secretion. EcoSal Plus, 8(2), 10.1128/ecosalplus.ESP-0040-2018. https://doi.org/10.1128/ecosalplus.ESP-0040-2018

  18. Liang, R., Zhou, J., & Liu, J. (2011). Construction of a bacterial assay for estrogen detection based on an estrogen-sensitive intein. Applied and environmental microbiology, 77(7), 2488–2495. https://doi.org/10.1128/AEM.02336-10

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