Team:BGU Israel/Solution

Overview

The processes of bioremediation, biosensors and bio-medicine often require the release of genetically modified organisms (GMO's) to the environment. After being released, these GMO's are no longer under direct control, therefore posing potential threats such as substituting the natural bacterial population, horizontal gene transfer, and lastly, creating public concern and opposition to the field of genetic engineering.

In order to overcome these issues, we are designing a genetic circuit that limits the lifetime of a bacterial population after it is released into the environment. Our ultimate goal is to allow the end user to program a GMO population to survive in the environment until it has completed its task, after which the entire population will disappear. Crucially, our mechanism will function independently, without any external intervention.

We are approaching this goal from two angles: "P.A.S.E 1" is based on the dilution of a vital component through cell division, while
"P.A.S.E 2" is based on the lifetime of an essential protein. The two methods utilize novel mechanisms such as a recombination cassette, the innovative use of an unnatural amino acid (UAA) in a biologic gate, and more. We also intend to introduce new biobricks that will be useful for a wide range of purposes, including bacterial genome incorporation parts and UAA incorporation machinery. This project will be the first modular, independent and generic bacterial control system.

We want our construct to pave the way to a safe synthetic revolution: one that will allow developers to innovate and create freely without arousing public
opposition or harming the environment.

P.A.S.E 1 (Programmable Autonomous Self Elimination 1)

Objective

To create a modular system to enable programming a cell to destroy itself after a predetermined amount of time.

Overview

The system consists primarily of two parts – a protective element, and a lethal element. The protective element is expressed by the cell only in the presence of a specific inducer, provided only in a controlled environment, the lab. As long as the protective element is present in the cell, it prevents expression of the lethal element. Through cell division, the protective element is diluted, and eventually, when its concentration falls under a critical threshold, it no longer protects the cell from the lethal element. This system is autonomous – unlike a kill-switch, it requires no external intervention to function after it has been released to the environment. Additionally, the system is intended to be programmable – through controlling different parameters during induction, it is possible to affect the concentration of the protective element before the cells are released. The higher the concentration upon release, the longer the cells would live.

Detailed design

The chassis used for the system is E. coli, strain BL-21. The functional system has two parts:

1. pUC57 plasmid

1. cI repressor gene (the protective element) - part BBa_K327018, edited to remove the LVA tail, which was originally meant to cause rapid degradation of the protein. Since the delay of the system greatly relies on the life time of cI, the tail was removed. The expression of this gene is regulated by the araC/lacI promoter.
2. lac/ara-1 promoter – part BBa_K354000. This promoter is activated by induction with IPTG and (but not necessarily) L - arabinose. [1] Using different variation of inducers (IPTG/Arabinsoe/IPTG+Arabinose) allows the user to adjust the induction strength, which controls the concentration of cI repressor in the cell. Higher concentration means longer life time of the population in the uncontrolled environment. Other ways to control induction strength have not yet been characterized - but our vision is that according to the user needs, different induction parameter or different promoters with different characteristics could be used to fine tune the life time of a population.
3. A constituently expressed resistance for Carbenicillin (to allow for selection).

2. A cassette which undergoes recombination into the bacterial chromosome

1. Coupled holin and lysozyme genes – part BBa_K112022, coding for the toxins that will eventually kill the cell. Their expression is regulated by the cI regulated promoter.
   
2. cI regulated promoter – par BBa_R0051. cI binding results in repression of transcription. When cI concentration is low enough, holin and lyzozyme genes are expressed.
   
3. A constituently expressed resistance for kanamycin (to allow for selection).
   

Why recombination? In our initial design, we intended to clone the toxin genes on a plasmid. However, one of the most important requirements of our system is that all cells must contain the toxin genes. In light of this requirement, plasmids pose a problem – their stability is influenced by the nature of the host cell, the type of plasmid and/or environmental conditions. Plasmid encoded properties may confer a selective advantage on the host cell but can be an energy drain due to replication and expression [2]. In our case – not only they confer no selective advantage, they carry a selective disadvantage! So, even if one cell would lose the toxin plasmid, it would not die, quickly giving rise to a population without the self-destruct system. In order to overcome this obstacle, we chose to use a cassette which undergoes recombination into the chromosome. The cell cannot lose the toxin gene, and is assured to pass it on to all daughter cells. This ensures the stability of the gene in the entire population.
Using recombination has another advantage for our system. Because the gene is located on the chromosome instead of plasmid, it has the lowest copy number possible. This theoretically makes the protective element effective at lower concentrations, which would allow for a longer life time of the population.

Assembling the system requires three transformations (although ultimately the cell will contain only one plasmid, and one addition to the chromosome):

1. pkd78 - make the cell recombination-ready: most bacteria are not readily transformable with linear DNA. One reason E. coli is not so transformable with linear DNA, is because of the presence of intracellular exonucleases that degrade linear DNA. In order to make the cells recombination-ready, we used the phage l Red recombinase. The recombination system we used was designed by Datsenko and Wanner: pkd78 is an easily cured, low copy number plasmid containing the phage l Red recombinase, which is synthesized under the control of arabinose. [3]
   
2. puc57 cI (Bba_K1223010) – insert the protective element gene.
   
3. Toxin cassette (Bba_K1223001) – insert the lethal element genes. Before transforming the cell with the cassette, in addition to following regular protocol for making competent cells, the culture has to be induced with arabinose and IPTG. Arabinose induces the recombination system, and IPTG induces the synthesis of cI, in order to prevent synthesis of the toxin after the transformation. After the transformation the culture is grown in 37oc curing the cell from pkd78.
   
   

[1] Lutz, R. & Bujard, H. (1997). “Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2regulatory elements”. Nucleic Acids Research, (25) 1203 – 1210.
[2] McLoughlin, A. J. (1994). “Plasmid stability and ecological competence in recombinant cultures”. Biotechnology Advances (12), 279–324.
[3] Datsenko K. A., Wanner, B.L. (2000). “One-step inactivation of chromosomal genes in Escherichia coliK-12 using PCR products”. PNAS (97), 6640-6645.

P.A.S.E 2 (Programmable Autonomous Self Elimination 2)

Objective

Creation of a modular system to program E.coli bacteria to destroy themselves after a predetermined amount of time.

Overview

In this system, we intentionally create a nonsense (TAG codon) mutation in one of E.coli’s essential genes – Tyrosyl tRNA synthetase (tyrS). Its location in the E.coli genome is 1,713,972 - 1,715,246 ( - ); 36.94 min; 1275 (bp) , 424 (aa). In addition, we add an inducible promoter, AraC/LacI, to regulate this gene. Unable to translate this gene, the bacteria will lose most of their protein synthesis capabilities, resulting first in proliferation arrest and eventually death. We then give the bacteria a specific machinery, which allows genetic code expansion and translation of the TAG stop codon, resulting in translation of the essential tyrS gene (See detailed design section). The genetic code expansion is possible only when all the machinery is in place and when a special “building block” (in the form of an unnatural amino acid), found only in the lab, is present. Moreover, the required machinery and the essential protein itself are translated exclusively when induced by compounds that are supplied only in the lab.

This mechanism is built to create a “logic AND gate” – the bacteria are dependent on two different inducers (Arabinose and IPTG/Lactose) to regulate transcription, and on special “building blocks” (an unnatural amino acid – UAA) to regulate translation. All three compounds are relatively rare in most natural environments. In the lab, the engineered cells are supplied with the building blocks, machinery and inducers, and thus synthesize the essential protein in large quantities. Once the bacteria are released from the lab, this protein naturally degrades over time. Since without the building blocks and machinery inducer the bacteria cannot synthesize more essential protein, once the protein reaches a critically low concentration the cell cannot sustain itself and dies. Currently we are working on creating one nonsense mutation in our target gene, but theoretically multiple nonsense mutations can be introduced, making the system even more fail-proof.

Like our first approach, this system is also autonomous – unlike a kill-switch, it requires no external intervention to function after it has been released to the environment. It can also be programmable - through controlling different parameters during induction, it is possible to affect the concentration of the essential protein before the cells are released. The higher the concentration upon release, the longer the cells would live. Additional control over the lifetime of the bacteria is possible through controlling the lifetime of the essential protein itself (N-terminus mutations), or choosing to work with different essential proteins with different lifetimes.

Detailed design

A short introduction to unnatural amino acids (UAA)

Other than a few exceptions, the genetic code is universal and encodes 20 amino acids, the building blocks of all proteins. Recently, it has become possible to incorporate man-made amino acids into proteins by using the degeneracy of the genetic code. The incorporation of unnatural amino acids (UAAs) enables the production of proteins with novel properties for new applications. The incorporation of UAAs uses the cell's natural protein synthesis mechanisms by introducing a pair of tRNAcua and tRNA-synthetase that do not cross-react with (i.e. are orthogonal to) endogenous natural amino acids/tRNA-synthetases.

A general approach of creating a system that will incorporate unnatural amino acids into proteins is described by L. Wang and P. G. Schultz [1]: the system that was developed consists of a tRNA and tRNA synthetase set that is orthogonal to the other amino acids and amino-acyl-tRNA-synthethases in the cell. The orthogonality of the system is achieved by using natural tRNA and tRNA-synthethase pair from another species, the archae Methanocaldococcus jannaschii. After mutating the tRNA anticodon sequence to C-U-A to suppress the amber stop codon (UAG), the tRNA and the synthethase were randomly mutated in chosen locations to produce a library for selection of the mutant with the desired properties. The selection of mutants was done by a series of negative and positive selection tests for orthogonally, ability to suppress the amber stop codon, and incorporation of unnatural amino acids into a protein. The gene of the target protein is site-specifically mutated to insert the amber stop codon (TAG) in the desired site for the incorporation of the UAA. The amber stop codon is used because it is the least used stop codon in the E. coli’s genetic code (only 314 sites are known) and therefore will minimally interfere with the synthesis of other proteins.

The chassis used for the system is E.coli, strain BL-21. The functional system has two parts:

1. UAA machinery (inserted into bacteria):

1. Orthogonal tRNAcua.
2. Acetyl-lysyl tRNA synthetase, regulated by LacI promoter.
3. A constituently expressed resistance for Chloramphenicol (for selection).
4. UAA: Acetyl-lysine.

2. A linear DNA cassette, which undergoes recombination into the bacterial chromosome:

1. Homology regions were designed so that the cassette will enter the chromosome in a specific site: it replaces the first 60 base pairs of the 20 N-ternimus codons of the Tyrosyl tRNA synthetase gene. The replaced sequence is the same as the original chromosomal sequence, with one change – the sixth amino acid code has been mutated to the amber stop codon (TAG). After the recombination, only cells with the UAA machinery (tRNA & tRNA synthetase couple) and UAA (Acetyl-lysine) supply can translate the Tyrosine tRNA Synthetase, an essential gene.
2. lac/ara-1 promoter – part BBa_K354000. Upstream of the homology site and the Tyrosine Synthetase gene, we added the lac/ara-1 promoter. This promoter is activated by induction with IPTG and (but not necessarily) L-arabinose.[2] Using different combinations of IPTG, Arabinsoe, or IPTG+Arabinose allows the user to adjust the induction strength, which controls the concentration of tyrosine synthetase in the cell. A higher concentration means a longer lifetime of the population in the uncontrolled environment. Other ways to control induction strength have not yet been characterized - but our vision is that different induction parameters or another promoter with different characteristics could be used to fine-tune the lifetime of a population according to the needs of the end user.
3. A constituently expressed resistance for kanamycin (for selection).

System Assembly

Assembling the system requires three transformations (although ultimately the cell will contain only one plasmid, and one change in the chromosome):

1. Transformation of pkd78 - makes the cell recombination-ready: most bacteria are not readily transformable with linear DNA. One reason E.coli is not easily transformable with linear DNA, is because of the presence of intracellular exonucleases that degrade linear DNA. In order to make the cells recombination-ready, we used the phage l Red recombinase. The recombination system used was designed by Datsenko and Wanner: pkd78 is an easily cured, low copy number plasmid containing the phage l Red recombinase, which upon induction with Arabinose makes the bacteria significantly susceptible to linear DNA cassette genome incorporation. [3] This pkd78 has a different antibiotic resistance than the one used in P.A.S.E 1.
2. Transformation of UAA machinery (tRNAcua and tRNA synthetase) into the bacterium on a protein expression plasmid (with high copy number).
3. Transformation of the P.A.S.E 2 cassette (Bba_K1223002) – adds a regulatory element (lacI/araC) to the chromosomal Tyrosine synthetase gene and inserts a nonsense mutation to it (TAG stop codon). Before transforming the cell with the cassette, in addition to following regular protocol for making competent cells, the culture has to be induced with arabinose, inducing the expression of the recombination proteins.
4. After genome cassette incorporation, the pkd78 is removed using heat shock or incubation in high temperatures (37-42 degrees Celsius).
5. The bacteria are grown in a rich medium containing two antibiotics (the antibiotic resistance is expressed from the UAA machinery plasmid and from the genome cassette insert), acetyl-lysine and inducers IPTG and Arabinose.
6. After the growth and accumulation of the essential recombinant protein, the medium is changed to a medium without antibiotics, inducers or acetyl-lysine.
7. Lastly the bacteria undergo (-80 degrees Celsius) freeze protocol and are ready for use.

[1] Wang, L. and P.G. Schultz, Expanding the genetic code. Chem Commun (Camb), 2002(1): p. 1-11.
[2] A. J. McLoughlin, Plasmid stability and ecological competence in recombinant cultures. Biotech. Advances 12(2), 279–324 (1994).
[3] K. A. Datsenko, B. L. Wanner, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. PNAS 97 (12), 6640-6645 (2000).