Team:St Andrews/Parts

Shinescreen: A Novel,Entirely Reef-Safe Probiotic Sunscreen.

Parts


Parts Overview

Introduction

Dreams of summer 2020, filled with the joys of repeated laboratory transformations and gel electrophoresis analysis were swiftly terminated in March as the COVID-19 pandemic stormed the U.K. and other parts of the globe. Any planned wet lab procedures were instantaneously cancelled and a ‘two-phase’ iGEM project was adopted thereafter.

The initial concept of a probiotic sunscreen utilising synthetic biology to provide sustainable sun protection was therefore not lost and the team began to speculate upon many different gene circuits designs our E.colii chassis would harness. From temperature sensing to novel two-plasmid kill switch mechanisms, the St Andrews iGEM team made use of the free research time usually occupied by lab work to synthesise a foolproof in silico circuit that would serve its prudent purpose on the surface of the human skin.

After many hours of research and design, the final parts required for the theoretical function of our organism were completed and uploaded to the registry (from BBa_K3634000 to BBa_K3634021). In summary, the gene circuit is split into two divisions: that of the shinorine-producing gene cluster (shinogen) as well as the kill switch component (thanogen). The two divisions are themselves split across two plasmids named pSB3B1-DOPH and pSB3E1-AN to contribute to the overall safety of the engineered organism.


Shinogen

The shinogen component consists of four conserved genes, taken from the cyanobacteria species Anabaena variabilis ATCC 29413, catalysing production of the UV-absorbing compound shinorine. Initially, the substrate sedoheptulose 7-phosphate is taken as an intermediate of the pentose phopshate pathway (PPP) and converted into (R)-demethyl-4-deoxygadusol by 3-dehydroquinate synthase (DHQS) (BBa_K3634000) catalysis. O-methyltransferase (O-MT) (BBa_K3634001) then catalyses the conversion of (R)/(S)-demethyl-4-deoxygadusol to 4-deoxygadusol (4-DG). Both BBa_K3634000 and BBa_K3634001 are codon optimised for E.coli to improve transcriptional efficiency and are contained on pSB3B1-DOPH.

The second cluster of genes for enzymes ATP-Grasp (ATPG) (BBa_K3634002) and non-ribosomal peptide synthetase (NRPS) (BBa_K3634003) are contained on plasmid pSB3E1-AN. These enzymes are responsible firstly for the conversion of 4-DG to the mycosporine-like amino acid (MAA) mycosporine glycine and then secondly to the final product shinorine. Separating the shinorine-producing gene cluster across two plasmids ensures organisms must take up both during horizontal gene transfer (HGT) to have any sort of conferred UV protection and survival advantage. The genes were selected across the two plasmids in this format purposefully as mycosporine glycine, an intermediate of the pathway, also has UV-absorbing potential at a different wavelength to that of shinorine. As with the first cluster of shinogen genes, BBa_K3634002 and BBa_K3634003 are both codons optimised for use in E.coli.



Thanogen

The thanogen component is vastly more complex than that of the shinogen and functions again across both pSB3B1-DOPH and pSB3E1-AN. Crucially, the thanogen system has two independent sensing modules for glucose and light (535nm specifically), functioning as both an AND and an OR gate simultaneously. The conditions for the thanogen component to function can be broken into four total scenarios:


  • The bacterium remains viable / In the presence of both light and glucose, expression of the antitoxin ccdA prevents any leaky ccdB toxin accumulation in the cell and represses an mf-Lon protease which would otherwise target lacI via a degradation tag and lead to expression of the R.CviJI endonuclease for cell destruction.
  • The bacterium remains in stasis and some nuclease activity is observed / In the presence of light and no glucose, expression of the antitoxin ccdA still occurs however expression of ccdB is now promoted. Intracellular ccdA:ccdB ratios change to favour ccdB concentrations and as ccdA is unstable, intracellular ccdB inhibits DNA gyrase and prevents cell growth. Some mf-Lon protease is expressed however due to the presence of ccdA, production of this enzyme is minimised so cellular destruction by R.CviJI endonuclease is limited.
  • Some bacteria remain in stasis and nuclease activity is observed / In the presence of no light and glucose, a small amount of leaky expression of the antitoxin will occur. CcdB expression is also leaky and not promoted therefore neutralisation of ccdB by ccdA will still occur to some extent. As the half-life of ccdA is shorter than ccdB, some stasis will occur but very little repression of mf-Lon protease expression will be observed - R.CviJI endonuclease activity is therefore expected.
  • The bacterium remains in stasis and nuclease activity is observed / In the absence of both light and glucose, a small amount of leaky expression of the antitoxin will occur. CcdB expression is promoted however and thus ccdA:ccdB ratios are greatly skewed to favour bacterial stasis. Limited repression of mf-Lon protease will also be observed thus lacI will be degraded by mf-Lon protease by the associated degradation tag and R.CviJI endonuclease expression will be enabled.

As with the shinogen component, the two-plasmid system functions in the thanogen to kill any bacteria that have obtained an individual plasmid alone by HGT. If pSB3B1-DOPH is taken up independently, expression of R.CviJI will not be sufficiently repressed by lacI due to its gene existing on pSB3E1-AN, resulting in DNA fragmentation and cell death. If pSB3E1-AN is taken up independently, leaky expression of ccdB will result in bacterial stasis and inhibition of DNA gyrase. The effect of ccdB can be rapidly reversed in the presence of ccdA and as ccdB also takes a while to accumulate in the cell, temporary factors such as shade and cloud cover will not affect bacterial viability when applied to the skin.

The following composite parts are the essential components of the kill switch mechanism:

pSB3B1-DOPH


Figure 1. Structure of the pSB3B1-DOPH Plasmid. Main genes labelled in maroon. Terminator in white. Ribosome in grey. Operator in turquoise. CAP-binding site in blue.


pSB3E1-AN


Figure 2. Structure of the pSB3E1-AN Plasmid. Main genes labelled in maroon. Terminator in white. Ribosome in grey.


PCB Chromophore and Membrane-associated Histidine Kinase ccaS

On top of the systems mentioned above, an essential chromophore component is also required for the light sensing system to function correctly as part of the thanogen. Part BBa_K3634009, present on pSB3E1-AN, describes the action of the photosystem in detail, made up of the membrane-associated histidine kinase ccaS (BBa_K3634006), heme oxygenase (ho1) (BBa_K3634007) and phycocyanobilin:ferredoxin oxidoreductase (pcyA) (BBa_K3634008). Ultimately, heme oxygenase and phycocyanobilin:ferredoxin oxidoreductase converts heme into the chromophore phycocyanobilin (PCB) which then binds to ccaS to impart reversible photoactivation.

Photoactivation of ccaS then allows phosphotransfer to ccaR present on pSB3B1-DOPH where expression of the output gene then follows. As with the shinogen, the chromophore component is also codon optimised for E.coli and is split from ccaR across two plasmids in part to increase maximal ccdA expression (Tabor et al., 2011) but also as a necessary biosafety mechanism. The optimised sequences are again characterised below:




Basic Parts

Below is a summary of all the basic parts created in silico throughout the experiment. All parts are either native to or optimised for usage in E.coli in order to improve gene expression efficiency. Combined as composite parts in plasmids pSB3B1-DOPH and pSB3E1-AN, the inserts will be used in phase II of the project to generate the UV-absorbing compound shinorine and through a novel kill switch method and mechanism, demonstrate an improvement upon the biosafety of previous synthetic biology applications.


Part Number Part Name Description Reference
BBa_K3634000 3-Dehydroquinate Synthase (DHQS) Codon optimised 3-dehydroquinate synthase (DHQS) CDS for use in E.coli as part of the shinorine synthesis gene cluster. The original gene for DHQS (Ava_3858) can be taken from the cyanobacteria species Anabaena variabilis ATCC 29413. The enzyme catalyses the conversion of sedoheptulose 7-phosphate, an intermediate of the pentose phosphate pathway (PPP), to (R)-demethyl-4-deoxygadusol. Minnesota iGEM 2012 - http://parts.igem.org/Part:BBa_K814000
BBa_K3634001 O-methyltransferase (O-MT) Codon optimised O-methyltransferase (O-MT) CDS for use in E.coli as part of the shinorine synthesis gene cluster. The original gene for O-MT (Ava_3857) can be taken from the cyanobacteria species Anabaena variabilis ATCC 29413. The enzyme catalyses the conversion of (R)/(S)-demethyl-4-deoxygadusol, the product of the first step of the shinorine pathway, to 4-deoxygadusol. Minnesota iGEM 2012 - http://parts.igem.org/Part:BBa_K814002
BBa_K3634002 ATP-Grasp (ATPG) Codon optimised adenosine triphosphate (ATP)-grasp (ATPG) CDS for use in E.coli as part of the shinorine synthesis gene cluster. The original gene for ATPG (Ava_3856) can be taken from the cyanobacteria species Anabaena variabilis ATCC 29413. The enzyme catalyses the conversion of 4-deoxygadusol (4-DG), produced by O-MT in the previous step of the reaction, to the mycosporine-like amino acid (MAA) mycosporine glycine. ATPG is proposed to phosphorylate 4-DG before conjugate addition occurs at the cyclohexanone ring by the nitrogen of glycine. Minnesota iGEM 2012 - http://parts.igem.org/Part:BBa_K814001
Balskus, E.P, Walsh, C.T. 2010. The Genetic and Molecular Basis for Sunscreen Biosynthesis in Cyanobacteria. Science. 329 (5999): p1653-1656. DOI: 10.1126/science.1193637
BBa_K3634003 Nonribosomal Peptide Synthetase (NRPS) Codon optimised nonribosomal peptide synthetase (NRPS) CDS for use in E.coli as the final part of the shinorine synthesis gene cluster. The original gene for NRPS (Ava_3855) can be taken from the cyanobacteria species Anabaena variabilis ATCC 29413. The enzyme catalyses the conversion of mycosporine glycine to the final product shinorine. NRPS has three known domains: an adenylation domain, a thiolation domain and a thioesterase domain. Initially, a serine amino acid is converted into an acyl adenylate species which is then later attacked by a serine residue present on the thiolation domain of NRPS. Adenosine monophosphate is then lost from the intermediate followed by imine formation, thought to occur through an enol ester intermediate and an O-N rearrangement by conjugate addition of the serine nitrogen to the cyclohexenimine ring. Minnesota iGEM 2012 - http://parts.igem.org/Part:BBa_K814003
Balskus, E.P, Walsh, C.T. 2010. The Genetic and Molecular Basis for Sunscreen Biosynthesis in Cyanobacteria. Science. 329 (5999): p1653-1656. DOI: 10.1126/science.1193637
BBa_K3634006 ccaS The membrane-associated histidine kinase ccaS is part of the two-component system (TCS) involved in the eventual transcriptional output of an adjacent phycobilisome-related gene (cpcG2) in response to green light of wavelength 535nm. The system is native to Synechocystis sp. PCC6803 but has been successfully expressed in E.coli (Hirose et al. 2008) and has been further used in multichromatic control of gene expression (Tabor et al. 2011). CcaS, alongside the response regulator ccaR, functions as a photoreversible switch between green (535nm) and red (672nm) light by regulation of the output promoter PcpcG2. Within the N-terminal GAF domain of ccaS, the blue phycobilin chromophore phycocyanobilin (PCB) binds to a conserved cysteine residue, imparting reversible photoactivation of signalling activity. Absorption of green light increases the rate of ccaS autophosphorylation, phosphotransfer to ccaR and transcription from PcpcG2. Absorption of red light by ccaS is shown to reverse the process and therefore reduce expression of the output gene. CcaS is also shown to be inactive in the dark. Hirose Y., Shimada T., Narikawa R., Katayama M., Ikeuchi M. 2008. Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. PNAS. 105(28): p9528-9533. DOI: 10.1073/pnas.0801826105
Tabor J.J., Levskaya A., Voigt C.A. 2011. Multichromatic control of gene expression in Escherichia coli. J Mol Biol. 405(2): p315–324. DOI: 10.1016/j.jmb.2010.10.038
BBa_K3634007 Heme Oxygenase (ho1) Heme oxygenase (ho1) is the first of two required genes for the conversion of heme into the blue phycobilin phycocyanobilin (PCB), a chromophore required for activation of some two component light-sensing systems (TCS) such as the ccaS/ccaR system in Synechocystis sp. PCC 6803. In the presence of oxygen, heme oxygenase catalyses the opening of the heme ring, releasing iron and generating biliverdin IXα which is then reduced by phycocyanobilin ferredoxin oxidoreductase (pcyA) to produce PCB. PCB can then bind to the appropriate membrane associated histidine kinase of the TCS allowing activation and expression of the output gene in the presence of an activating wavelength of light. Tabor J.J., Levskaya A., Voigt C.A. 2011. Multichromatic control of gene expression in Escherichia coli. J Mol Biol. 405(2): p315–324. DOI: 10.1016/j.jmb.2010.10.038
UTAustin iGEM 2004 - http://parts.igem.org/Part:BBa_I15008
BBa_K3634008 Phycocyanobilin:Ferredoxin Oxidoreductase (pcyA) The gene pcyA codes for the functional phycocyanobilin:ferredoxin oxidoreductase protein, the second enzyme required for the conversion of heme to the blue phycobilin phycocyanobilin (PCB); a chromophore required for activation of some two component light-sensing systems (TCS) such as the ccaS/ccaR system in Synechocystis sp. PCC 6803. The protein catalyses the four-electron reduction of biliverdin IXα, previously produced from heme by heme oxygenase, to PCB, which then binds to the appropriate membrane associated histidine kinase of the TCS allowing activation and expression of the output gene in the presence of an activating wavelength of light. Tabor J.J., Levskaya A., Voigt C.A. 2011. Multichromatic control of gene expression in Escherichia coli. J Mol Biol. 405(2): p315–324. DOI: 10.1016/j.jmb.2010.10.038
UTAustin iGEM 2004 - http://parts.igem.org/Part:BBa_I15009
BBa_K3634010 LacO- & LacP The lac operon found in E.coli consists of the three lactose metabolising genes lacZ, lacY and lacA which when expressed, allow the bacteria to use the sugar as a source of energy. The initial regulatory mechanisms in the pathway were outlined by Jacob and Monod in 1961, where the topic of inducible and repressible enzyme systems was discussed. In this system, the transcriptional repressor is a protein known as Lac I which binds to DNA at various operator sequences (termed O1, O2 and O3) which exist both upstream and downstream of the transcriptional start site (TSS). Interaction between the Lac I and operator sequences reduces transcription of the downstream lactose metabolising genes unless relieved by the lactose isomer allolactose. In the absence of Lac I, transcription is constitutive and can be further activated by the catabolite activator protein (CAP), with binding site upstream of the promoter sequence. Oehler et al. (1990) mutated each individual operator sequence respectively and then determined the effect of repression by Lac I to which they found mutation in O1 (downstream of the promoter) sufficient to lose almost all total repression. Mutation of O2 and O3 further decreased repression by Lac I 70 fold. Here, the St Andrews iGEM team 2020 aimed to utilise these findings to create a regulatory region solely under the control of glucose concentration to allow expression of the toxin ccdB. Bernard P., Couturier M. 1992. Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. J. Mol. Biol. 226: p735–745.
Vandervelde A., Drobnak I., Hadži S., Sterckx Y.GJ., Welte T., Greve. H.D., Charlier D., Efremov R., Loris R., Lah J. 2017. Molecular mechanism governing ratio-dependent transcription regulation in the ccdAB operon. NAR. 45(6): p2937-2950. DOI: 10.1093/nar/gkx108
BBa_K3634011 ccdB The ccdAB toxin-antitoxin (TA) module is a type II TA module where the toxic ccdB protein, poisons the enzyme DNA gyrase, required for negative supercoiling of DNA (Bernard and Couturier, 1992). Through ccdB-gyrase complex formation, DNA cleavage results as well as inhibition of transcription by the formation of RNA polymerase roadblocks. The activity of the unstable ccdA antitoxin separates the ccdB-gyrase complex if present (Vandervelde et al, 2017). Jacob F., Monod J. 1961. Genetic Regulatory Mechanisms in the Synthesis of Proteins. J. Mol. Biol. 3: p818-356.
Gilbert W., Maxam A. 1973. The Nucleotide Sequence of the lac Operator. Proc. Nat. Acad. Sci. USA. 70(12): p3581-3584.
Oehler S., Eismann E.R., Krämer H., Müller-Hill B. 1990. The three operators of the lac operon cooperate in repression. EMBO J. 9(v): p973-979.
BBa_K3634013 ccdAB Promoter & Operator Expression of ccdA and ccdB within the type II TA module is self-regulated by low specificity and affinity of ccdA for individual binding sites of the regulatory region upstream of both ccdA and ccdB genes. This 113bp ccdAB promoter/operator sequence extends into the first ccdA gene and is suspected to have a total of 8 ccdA operator binding sites (Tam & Kline, 1989). The antitoxin binds the operator DNA sites, with some sites overlapping the promoter, functioning as a repressor of ccdAB transcription. The toxin then functions as a co-repressor or de-repressor depending on the molar T:A ratio (Vandervelde et al., 2017). Tam J.E., Kline B.C. 1989. Control of the ccd operon in plasmid F. J. Bacteriol. 171: p2353–2360.
Vandervelde A., Drobnak I., Hadži S., Sterckx Y.GJ., Welte T., Greve. H.D., Charlier D., Efremov R., Loris R., Lah J. 2017. Molecular mechanism governing ratio-dependent transcription regulation in the ccdAB operon. NAR. 45(6): p2937-2950.
BBa_K3634015 LacI + mf-Lon Degradation Tag BBa_K3634015 is a fusion between the lacI gene and the mf-Lon degradation tag, a specific sequence recognised by the mf-Lon protease which allows for rapid endogenous breakdown of the protein to which it is attached. The degradation tag used here is the strongest characterised sequence for the specific protease (BBa_K2333001) and will allow for efficient removal of Lac I from the cell when mf-Lon protease is expressed (BBa_K3634014). Jacob F., Monod J. 1961. Genetic Regulatory Mechanisms in the Synthesis of Proteins. J. Mol. Biol. 3: p818-356.
Oehler S., Eismann E.R., Krämer H., Müller-Hill B. 1990. The three operators of the lac operon cooperate in repression. EMBO J. 9(v): p973-979.
BBa_K3634017 ccaR The response regulator ccaR is part of the two-component system (TCS) involved in the eventual transcriptional output of an adjacent phycobilisome-related gene (cpcG2) in response to green light of wavelength 535nm. The system is native to Synechocystis sp. PCC6803 but has been successfully expressed in E.coli (Hirose et al. 2008) and has been further used in multichromatic control of gene expression (Tabor et al. 2011). CcaR, alongside the membrane-associated histidine kinase ccaS, functions as a photoreversible switch between green (535nm) and red (672nm) light by regulation of the output promoter PcpcG2. Within the N-terminal GAF domain of ccaS, the blue phycobilin chromophore phycocyanobilin (PCB) binds to a conserved cysteine residue, imparting reversible photoactivation of signalling activity. Absorption of green light increases the rate of ccaS autophosphorylation and phosphotransfer to ccaR. Once phosphotransfer has occurred, ccaR binds to an operator site within the sequence of the output promoter PcpcG2. Transcription of the output gene is then activated. Hirose Y., Shimada T., Narikawa R., Katayama M., Ikeuchi M. 2008. Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. PNAS. 105(28): p9528-9533. DOI: 10.1073􏰃/pnas.0801826105
Tabor J.J., Levskaya A., Voigt C.A. 2011. Multichromatic control of gene expression in Escherichia coli. J Mol Biol. 405(2): p315–324. DOI: 10.1016/j.jmb.2010.10.038
Schmidl S.R., Sheth R.U., Wu A., Tabor J.J. 2014. Refactoring and Optimization of Light-Switchable Escherichia coli Two-Component Systems. ACS Synth Biol. 3: p820-831. DOI: 10.1021/sb500273n
BBa_K3634018 PL8-UV5 The lac operon found in E.coli consists of the three lactose metabolising genes lacZ, lacY and lacA which when expressed, allow the bacteria to use the sugar as a source of energy. The initial regulatory mechanisms in the pathway were outlined by Jacob and Monod in 1961, where the topic of inducible and repressible enzyme systems was discussed. In this system, the transcriptional repressor is a protein known as Lac I which binds to DNA at various operator sequences (termed O1, O2 and O3) which exist both upstream and downstream of the transcriptional start site (TSS). Interaction between the Lac I and operator sequences reduces transcription of the downstream lactose metabolising genes unless relieved by the lactose isomer allolactose. In the absence of Lac I, transcription is constitutive and can be further activated by the catabolite activator protein (CAP), with binding site upstream of the promoter sequence. Reznikoff et al. (1978) mutated the regulatory region in question at three different sites. Within the CAP binding site, bases -66 (G) and -55 (C) of the wt binding region were substituted with A and T respectively to prevent binding of the CAP protein at low glucose concentrations. The wt -10 promoter sequence was also mutated from TATGTT to TATAAT in order to allow σ factor (RpoD) to bind without relying on further activation by the CAP protein. As a result of these mutations, gene expression mediated by the PL8-UV5 promoter will be independently regulated by intracellular concentrations of the lacI repressor as all CAP-associated regulation has been removed. Jacob F., Monod J. 1961. Genetic Regulatory Mechanisms in the Synthesis of Proteins. J. Mol. Biol. 3: p818-356.
Reznikoff W. S., Abelson J. N. 1978. The lac promoter. The operon. 7: p221-243. DOI: 10.1101/0.221-243
Hirschel B.J., Shen V., Schlessinger D. 1980. Lactose Operon Transcription from Wild-Type and L8-UV5 lac Promoters in Escherichia coli Treated with Chloramphenicol. J. Bacteriol. 143(3): p1534-1537.
BBa_K3634020 R.CviJI Endonuclease + ssrA Degradation Tag The restriction endonuclease CviJI (also known as R.CviJI) is taken natively from the Chlorella virus IL-3A, a double-stranded DNA phycodnavirus that infects unicellular, eukaryotic Chlorella-like green algae. As well as being previously expressed in E.coli by Skowron et al. (1995) and Swaminathan et al. (1996), the restriction endonuclease is also used commercially and is available via NEB as CviKI-1. The enzyme cuts at RG/CY sites (where R = purines, Y = pyrimidines) in the presence of Mg2+. With the addition of ATP, R.CviJI (now R.CviJI*) cleaves at additional restriction sites RG/CN and YG/CY (where N = any nucleotide) but not YG/CR. Both enzymes cleave DNA frequently and therefore possess a variety of functions such as generating numerous sequence-specific oligonucleotides. The sequence to be used in this part is 278 amino acids in length and does not exhibit additional R.CviJI* activity. The 144-235 amino acid region is also suggested to have a recognition/catalytic domain. R.CviJI will be fused to the ssrA degradation tag AANDENYADAS to prevent plasmid destruction as a result of leaky expression only. Lon protease, native to E.coli, will recognise and degrade the fusion construct. The TAG stop codon of the R.CviJI gene was removed and replaced with the AANDENYADAS sequence. TAATAA was then added to the 3' end of the the ssrA to terminate translation. Skowron PM, Swaminathan N, McMaster K, George D, Van Etten JL, Mead DA. 1995. Cloning and applications of the two/three-base restriction endonuclease R.CviJI from IL-3A virus-infected Chlorella. Gene. 157: p37-41. DOI: 10.1016/0378-1119(94)00564-9.
Swaminathan N, Mead DA, McMaster K, George D, Van Etten JL, Skowron PM. 1996. Molecular cloning of the three base restriction endonuclease R.CviJI from eukaryotic Chlorella virus IL-3A. Nucleic Acids Res. 24: p2463-2469. DOI: 10.1093/nar/24.13.2463.


Composite Parts

Across pSB3B1-DOPH and pSB3E1-AN, a total of eight composite parts have been constructed, with five on the first plasmid and three on the latter. As previously discussed on the parts overview, the shinogen component is split into two separate composite parts; BBa_K3634005 on pSB3E1-AN and BBa_K3634004 on pSB3E1-AN. The thanogen component has four composite parts on pSB3E1-AN and two composite parts on pSB3B1-DOPH. All composite parts and their respective functions are better described individually but are listed in table format along with their basic counterparts within ‘Part Collection’.

Shinogen

BBa_K3634004 (4-DG Pathway)

The following part describes the constitutive expression of the first two enzymes of the shinorine production pathway, optimised for use in E.coli. Within the composite part, biobricks BBa_K3634000 (DHQS) and BBa_K3634001 (O-MT) are responsible for converting sedoheptulose 7-phosphate to 4-deoxygadusol. Once produced, 4-deoxygadusol will then be converted by BBa_K3634005 (ATPG and NRPS composite, present on 'pSB3E1-AN') to the final product of the pathway, shinorine. The shinorine production pathway is separated in this way as a biosafety mechanism so that UV resistance is not conferred in a bacteria which has lost or gained pSB3E1-AN/pSB3B1-DOPH alone. As NRPS is determined to be 'rate-limiting' with respect to the pathway, pSB3E1-AN will ideally be placed at a higher copy number to pSB3B1-DOPH to ensure 1:1 stoichiometry of reactants.

For our bacteria to provide maximum expression of the UV absorbing compound shinorine, the strong constitutive promoter BBa_J23119 was selected from the Anderson promoter catalogue alongside RBS BBa_B0034 inserted between each ORF. These parts are a preliminary choice based off previous experimental expression data (Berkeley iGEM, 2006). The commonly used double terminator BBa_B0015 will ensure reliable release of the new mRNA strand.


Design Notes

Due to the two-phase nature of our project, the selected promoter and RBS used may be subject to change after wet lab application in 2021 to maximise shinorine production. Golden gate assembly methods will facilitate this optimisation if necessary. It is also worth noting that the third enzyme (ATPG) may be included on pSB3E1-AN with NRPS instead of on pSB3B1-DOPH such that mycosporine glycine, which also absorbs UV radiation to a certain degree, is not produced in a bacteria which by chance has taken up pSB3B1-DOPH and would thereafter exploit UV resistance to confer a survival advantage in the environment.

Source

The initial genomic sequence came from Anabaena variabilis ATCC 29413 which can be obtained from parts BBa_K814000, BBa_K814002 and K8140001 (Minnesota iGEM, 2012) before being optimised using the IDT optimisation tool. The promoter and RBS were selected from each respective Anderson catalogue based on their relative strength. The terminator BBa_B0015 sequence can also be taken from the iGEM registry.


BBa_K3634005 (ATPG and NRPS Composite)

The following part describes the constitutive expression of the final two enzymes of the shinorine production pathway, optimised for use in E.coli. Within the composite part, biobrick BBa_K3634002 (ATPG) and BBa_K3634003 (NRPS) are responsible for converting 4-deoxygadusol (4-DG) to the final product shinorine. Once produced, this mycosporine-like amino acid can absorb UV radiation of wavelength ~333nm. The composite part described will be present on 'pSB3E1-AN', separate to the previous enzymes responsible for 4-deoxygadusol production which are maintained on ' pSB3B1-DOPH'. By this arrangement, biosafety of our gene circuit will be ensured such that UV resistance is not conferred in a bacteria which has lost or gained pSB3E1-AN / pSB3B1-DOPH alone. As NRPS is determined to be 'rate-limiting' with respect to the pathway, pSB3E1-AN will ideally be placed at a higher copy number to pSB3B1-DOPH to ensure 1:1 stoichiometry of reactants.

The enzyme ATPG was included on pSB3E1-AN with NRPS instead of on pSB3B1-DOPH with DHQS and O-MT as mycosporine glycine, an intermediate which also absorbs UV radiation, would have been produced. If a bacteria by chance was transformed with this three enzyme pSB3B1-DOPH variant, the organism would be UV resistant to a certain degree which therefore would confer a survival advantage in the environment.

For our bacteria to provide maximum expression of the UV absorbing compound shinorine, the strong constitutive promoter BBa_J23119 was selected from the Anderson promoter catalogue alongside RBS BBa_B0034. These parts are a preliminary choice based off previous experimental expression data (Berkeley iGEM, 2006). The commonly used double terminator BBa_B0015 will also ensure reliable termination.


Design Notes

Due to the two-phase nature of our project, the selected promoter and RBS used may be subject to change after wet lab application in 2021 to maximise shinorine production. Golden gate assembly methods will facilitate this optimisation if necessary. It may also be required to include more than one single promoter for adequate expression of NRPS compared to the other enzymes involved in the shinorine pathway.

Source

The initial genomic sequence came from Anabaena variabilis ATCC 29413 which can be obtained from part K8140003 (Minnesota iGEM, 2012) before being optimised using the IDT optimisation tool. The promoter and RBS were selected from each respective Anderson catalogue based on their relative strength. The terminator BBa_B0015 sequence can also be taken from the iGEM registry.


Thanogen

BBa_K3634009 (ccaS & ho1 & pcyA)

The composite part is present on pSB3E1-AN and is required for part BBa_K3634019 to function on pSB3B1-DOPH.

The membrane-associated histidine kinase ccaS is part of the two-component system (TCS) involved in the eventual transcriptional output of an adjacent phycobilisome-related gene (cpcG2). The system is native to Synechocystis sp. PCC6803 but has been successfully expressed in E.coli (Hirose et al. 2008) and has been further used in multichromatic control of gene expression (Tabor et al. 2011).

CcaS, alongside the response regulator ccaR, functions as a photoreversible switch between green (535nm) and red (672nm) light by regulation of the output promoter PcpcG2. Within the N-terminal GAF domain of ccaS, the blue phycobilin chromophore phycocyanobilin (PCB) binds to a conserved cysteine residue, imparting reversible photoactivation of signalling activity. Absorption of green light increases the rate of ccaS autophosphorylation, phosphotransfer to ccaR and transcription from PcpcG2. Absorption of red light by ccaS is shown to reverse the process and therefore reduce expression of the output gene. CcaS is also shown to be inactive in the dark. The required PCB chromophore is produced from heme by the following two enzymes.

In the presence of oxygen, heme oxygenase (ho1) catalyses the opening of the heme ring, releasing iron and generating biliverdin IXα. The second enzyme, phycocyanobilin:ferredoxin oxidoreductase (pcyA), then catalyses the four-electron reduction of biliverdin IXα to PCB. PCB then binds to the N-terminal GAF domain of ccaS, allowing for transcriptional output of the gene of interest.

BBa_K3634006, BBa_K3634007 and BBa_K3634008 are combined here as a composite part with the associated regulatory regions used by Schmidl et al. (2014). The system they produced, termed CcaSR v2, uses two plasmids for constitutive expression of ccaS and ccaR, and optimised promoter and RBS combinations to maximise PCB production. The design will be recreated in composite BioBrick part form using E.coli codon optimised parts to further promote efficiency of the system.


Design Notes

Schmidl et al. (2014) used both promoter and RBS libraries to produce the system termed CcaSR v2. Two plasmids are used for constitutive expression of ccaS and ccaR as this was found to increase output of sfgfp, measured quantitatively. Promoter and RBS combinations were also selected to maximise PCB production.

Source

The genomic sequences for all three CDS are IDT codon optimised for E.coli, taken from the native Synechocystis sp. PCC6803. Design of the composite part can be fully attributed to the work of Schmidl S.R., Sheth R.U., Wu A. and Tabor J.J. in the paper 'Refactoring and Optimization of Light-Switchable Escherichia coli Two-Component Systems'. Individual sequences for the parts can be found at BBa_K3634006, BBa_K3634007 and BBa_K3634008.


BBa_K3634019 (ccaR-Mediated ccdA Expression System)

The composite part is present on pSB3B1-DOPH and functions as an output to green light sensing by expressing ccdA - the antitoxin to ccdB.

The response regulator ccaR is part of the two-component system (TCS) involved in the eventual transcriptional output of an adjacent phycobilisome-related gene (cpcG2) in response to green light of wavelength 535nm. The system is native to Synechocystis sp. PCC6803 but has been successfully expressed in E.coli (Hirose et al. 2008) and has been further used in multichromatic control of gene expression (Tabor et al. 2011).

CcaR, alongside the membrane-associated histidine kinase ccaS, functions as a photoreversible switch between green (535nm) and red (672nm) light by regulation of the output promoter PcpcG2. Within the N-terminal GAF domain of ccaS, the blue phycobilin chromophore phycocyanobilin (PCB) binds to a conserved cysteine residue, imparting reversible photoactivation of signalling activity. Absorption of green light increases the rate of ccaS autophosphorylation and phosphotransfer to ccaR. Once phosphotransfer has occurred, ccaR binds to an operator site within the sequence of the output promoter PcpcG2. Transcription of the output gene is then activated.

The natural output gene cpcG2 has in the past been replaced with sfgfp in order to quantitatively measure the output of the light-sensing system. Replacing this gene with another output is therefore theoretically feasible. Here, the St Andrews iGEM team of 2020 aims to control the expression of the antitoxin ccdA in response to green light. Under activating conditions, expression of ccdA will be maximised whilst also minimising leaky expression from PcpcG2 by using a truncated version of the promoter (PcpcG2-172), characterised by Schmidl et al. (2014).

By regulating expression of ccdA, the dual functionality of the antitoxin can be utilised to prevent ccdB-gyrase formation (where ccdB is previously expressed in BBa_K3634012) and allow for precise transcriptional control of a desired output gene (see BBa_K3634014) (Vandervelde et al., 2017).


Design Notes

In the initial sequence obtained from Tabor's pJT122 plasmid, an illegal EcoR1 site is found. Uppsala-Sweden iGEM 2011 removed the site by carrying out point mutagenesis of A4C and A6C to obtain the part BBa_K592002. The part sequence was then subject to codon optimisation using the IDT codon optimisation tool to allow optimum expression of ccaR in the E.coli chassis. No new illegal restriction sites were introduced by this step. PcpcG2-172 sequence remained unchanged from pJT119 and the same for ccdA. All other promoters/RBS were chosen from the respective Anderson catalogues from the iGEM registry based off the design of pSR58.6 shown by Ong and Tabor (2018).

Source

ccaR & PcpcG2-172 can be found in the genome of Synechocystis PCC 6803. The sequences can be obtained from BBa_K592002 (Uppsala-Sweden iGEM, 2011 - initially from Tabor's pJT122 plasmid) and BBa_K2012015 (Huazhong Agricultural University iGEM, 2016 - initially from Tabor's pJT119 plasmid) respectively. The part sequence of BBa_K592002 was then fully optimised for our chosen chassis organism, E.coli, using the IDT codon optimisation tool. The ccdA antitoxin is found natively in E.coli with sequence obtained from the pCoo plasmid (NCBI Accession Number: NC_007635.1). This was confirmed with sequence from NCBI Accession Number: NC_019000.


BBa_K3634012 (Glucose-Mediated Death Sensor)

The composite part is present on pSB3E1-AN. The 'glucose-mediated death sensor' is a vital part of the St Andrews iGEM 2020 kill switch as when glucose is absent, greater expression of ccdB will overcome intracellular ccdA concentrations causing transcriptional inhibition and DNA cleavage as previously discussed. Expression of ccdB will also relieve the associated ccdAB promoter from ccdA binding which as a consequence, will allow for cviJI endonuclease expression to chew up the integrated plasmid constructs and target restriction sites within the genome.


lacO- + lacP

The lac operon found in E.coli consists of the three lactose metabolising genes lacZ, lacY and lacA which when expressed, allow the bacteria to use the sugar as a source of energy. The initial regulatory mechanisms in the pathway were outlined by Jacob and Monod in 1961, where the topic of inducible and repressible enzyme systems was discussed. In this system, the transcriptional repressor is a protein known as Lac I which binds to DNA at various operator sequences (termed O1, O2 and O3) which exist both upstream and downstream of the transcriptional start site (TSS). Interaction between the Lac I and operator sequences reduces transcription of the downstream lactose metabolising genes unless relieved by the lactose isomer allolactose. In the absence of Lac I, transcription is constitutive and can be further activated by the catabolite activator protein (CAP), with binding site upstream of the promoter sequence.

Oehler et al. (1990) mutated each individual operator sequence respectively and then determined the effect of repression by Lac I to which they found mutation in O1 (downstream of the promoter) sufficient to lose almost all total repression. Mutation of O2 and O3 further decreased repression by Lac I 70 fold. Here, the St Andrews iGEM team 2020 aimed to utilise these findings to create a regulatory region solely under the control of glucose concentration to allow expression of the toxin ccdB.


ccdB

The ccdAB toxin-antitoxin (TA) module is a type II TA module where the toxic ccdB protein, poisons the enzyme DNA gyrase, required for negative supercoiling of DNA (Bernard and Couturier, 1992). Through ccdB-gyrase complex formation, DNA cleavage results as well as inhibition of transcription by the formation of RNA polymerase roadblocks. The activity of the unstable ccdA antitoxin separates the ccdB-gyrase complex if present (Vandervelde et al, 2017).


Design Notes

The sequence for O2 was found 401bp downstream of O1, included in the lacZ gene, and was therefore not included as lacZ, lacY and lacA were later replaced by ccdB. The O3 sequence was found to have a 13bp overlap with the CAP binding site therefore was not mutated as this may have affected the CAP binding affinity. The sequence was truncated however where the CAP binding site and O3 5' sequence did not overlap. This was proposed to reduce binding affinity of the repressor. Three mutations were made to the O1 sequence: g91t, t98g and c101g. The native 6bp RBS downstream of lacO1 was also replaced with the weak RBS BBa_B0031 with spacer regions either side left as previous. The ccdB sequence was taken from BBa_K145151 and found to have an illegal BsaI restriction site at 222bp. The appropriate alteration was made to make the part RFC[1000] compatible: c219g (GTC (Val) to got to go (Val)). As native to E.coli, no codon optimisation step was required.

Source

The lac operon with the associated regulatory upstream components are found natively in E.coli. The initial sequence of the lacO1 was taken from Gilbert and Maxam (1973) and later confirmed by Oehler et al. (1990), where other sequences included in this part and appropriate mutations were gathered. The ccdB toxin is also found natively in E.coli and the sequence specified in BBa_K145151 by KULeuven iGEM (2008). This sequence was confirmed against pAG415 GPD-EGFP and pCMV SPORT6ccdB plasmids and in silico site-directed mutagenesis removed a BsaI site so that the part was RFC[1000] standard (BBa_K3634011).


BBa_K3634014 (ccdAB-Controlled mf-Lon Protease)

The composite part is present on pSB3E1-AN. As with BBa_K3634012, intracellular ccdAB concentrations tightly regulate the associated ccdAB promoter/operator sequence which in turn regulates the expression of the mf-Lon protease, required to degrade lacI to prevent lacI repression of R.CviJI endonuclease to result in cellular destruction.

Expression of ccdA and ccdB within the type II TA module is self-regulated by low specificity and affinity of ccdA for individual binding sites of the regulatory region upstream of both ccdA and ccdB genes. This 113bp ccdAB promoter/operator sequence extends into the first ccdA gene and is suspected to have a total of 8 ccdA operator binding sites (Tam & Kline, 1989). The antitoxin binds the operator DNA sites, with some sites overlapping the promoter, functioning as a repressor of ccdAB transcription. The toxin then functions as a co-repressor or de-repressor depending on the molar T:A ratio (Vandervelde et al., 2017).

At low T:A ratio, the operator is repressed however as the ratio is increased, repression is relieved by preferential formation of a V-shaped non-repressing heterohexamer (ccdB-ccdA-ccdB). Specifically, at the moment the molar ratio of T:A > 1, repression is rapidly lost (Vandervelde et al., 2017). Therefore the regulatory region, controlled by intracellular ccdAB concentrations, can be used for precise control of a desired output gene as shown in the native system.

Here, the output protein expressed will be mf-Lon protease (BBa_K2333011). mf-Lon protease is evolutionary related to the native Lon protease in E.coli and functions similarly in that recognition of a specific degradation tag allows for rapid endogenous breakdown of the protein to which it was attached. The two enzymes differ in their ability to recognise these specific tags; mf-Lon protease cannot recognise degradation tags associated to E.coli lon protease and vice versa. By the ability to alter the strength of signals associated with these degradation tags through sequence specific alterations (Collins et al., 2014), intracellular concentrations of proteins can be precisely modulated.


Design Notes

As aforementioned, the 113bp ccdAB promoter/operator region merges with the ccdA gene 87bp through the 5' end of the regulatory region. If included in full, all 8 ccdA binding sites would be part of the sequence allowing for maximum control efficiency of gene output (Vandervelde et al., 2017). However, as a RBS was predicted to be present before the ccdA gene, a truncated malfunctioning ccdA protein may well have been expressed. This would have resulted in an unwanted addition to cell metabolic burden and the truncated ccdA may have disrupted binding of functional ccdA to the operator sites.

As 5/8 ccdA binding sites remained unidentified in the 113bp region, it was predicted that removing the ccdA gene sequence would remove a maximum of 3 additional ccdA binding sites based on the observed length of the operator sequences and the spacer regions that existed between the three already identified regions. By the assumption that 5 binding sites would be present in the remaining 86bp promoter/operator region and comparison with Figure 4c of Vandervelde et al. (2017), it was concluded that the efficiency of repression by ccdA would remain high enough to warrant the use of the truncated promoter/operator region in our design.

With regard to the unknown RBS region, adding the weak BBa_B0031 RBS from the Anderson RBS catalogue on top of the pre-existing sequence would ensure sufficient expression of mf-Lon protease. The mf-Lon protease sequence itself was codon optimised for E. coli using the website www.jcat.de to improve expression efficiency in the chassis organism.

Source

The ccdAB operon is native to the F plasmid of E.coli. The specific sequence detailed for this part was obtained from Vandervelde et al. (2017) in 'Molecular Mechanisms Governing Ratio-Dependent Transcription Regulation in the CcdAB Operon'. The mf-Lon protease was obtained from BBa_K2333011 which was adapted from Gur & Sauer's 'Evolution of the ssrA degradation tag in Mycoplasma: specificity switch to a different protease' (2008). The part was also codon optimised for E.coli by William & Mary iGEM 2017 using the website www.jcat.de.


BBa_K3634016 (PlacIq & LacI Repressor + mf-Lon Degradation Tag)

The composite part is present on pSB3E1-AN. Here, alongside a more efficient promoter, lacI is attached to a mf-Lon protease degradation tag to allow for R.CviJI endonuclease expression and cellular destruction when intracellular ccdA concentrations are low (see above).

The lac operon found in E.coli consists of the three lactose metabolising genes lacZ, lacY and lacA which when expressed, allow the bacteria to use the sugar as a source of energy. The initial regulatory mechanisms in the pathway were outlined by Jacob and Monod in 1961, where the topic of inducible and repressible enzyme systems was discussed. In this system, the transcriptional repressor is a protein known as Lac I which binds to DNA at various operator sequences (termed O1, O2 and O3) which exist both upstream and downstream of the transcriptional start site (TSS). Interaction between the Lac I and operator sequences reduces transcription of the downstream lactose metabolising genes unless relieved by the lactose isomer allolactose. In the absence of Lac I, transcription is weakly constitutive and can be further activated by the catabolite activator protein (CAP), with binding site upstream of the promoter sequence (Oehler et al., 1990).

BBa_K3634015 is a fusion between the lacI gene and the mf-Lon degradation tag, a specific sequence recognised by the mf-Lon protease which allows for rapid endogenous breakdown of the protein to which it is attached. The degradation tag used here is the strongest characterised sequence for the specific protease (BBa_K2333001) and will allow for efficient removal of Lac I from the cell when mf-Lon protease is expressed (BBa_K3634014).

A modified version of the lacI promoter (lacIq - featuring an 'up' mutation 20bp into the sequence (-35 region: c -> t)) is also present in the composite part, allowing RNA polymerase to bind the promoter more tightly than the original which leads to a ten-fold increase in lacI expression and therefore a reduction in leaky expression from lacP (Calos, 1978)(Glascock & Weickert, 1998).


Design Notes

The sequence for lacI was taken from BBa_K1695000 with the 3' tga stop codon removed and the initiation start codon atg replaced with the native gtg start codon to improve protein expression efficiency. BBa_K2333001, the strong mf-Lon degradation tag taken from Mesoplasma florum, was then directly attached to the 3' end of the lacI gene to give the in silico fusion product.

Source

LacI alongside its promoter is found natively in E.coli. The initial sequence used for lacI was taken from BBa_K1695000 (see design notes) and the mutant promoter taken from BBa_K091111. The strong mf-Lon degradation tag is native to Mesoplasma florum and the sequence taken from BBa_K2333001.


BBa_K3634021 (LacI-Controlled CviJI Endonuclease + ssrA Degradation Tag)

The composite part is present on pSB3B1-DOPH. The restriction endonuclease CviJI moderating the destruction of cellular material will be regulated by an independent lacI-controlled promoter which itself is controlled overall by ccdA concentrations.

PL8-UV5

The lac operon found in E.coli consists of the three lactose metabolising genes lacZ, lacY and lacA which when expressed, allow the bacteria to use the sugar as a source of energy. The initial regulatory mechanisms in the pathway were outlined by Jacob and Monod in 1961, where the topic of inducible and repressible enzyme systems was discussed. In this system, the transcriptional repressor is a protein known as Lac I which binds to DNA at various operator sequences (termed O1, O2 and O3) which exist both upstream and downstream of the transcriptional start site (TSS). Interaction between the Lac I and operator sequences reduces transcription of the downstream lactose metabolising genes unless relieved by the lactose isomer allolactose. In the absence of Lac I, transcription is constitutive and can be further activated by the catabolite activator protein (CAP), with binding site upstream of the promoter sequence.

Reznikoff et al. (1978) mutated the regulatory region in question at three different sites. Within the CAP binding site, bases -66 (G) and -55 (C) of the wt binding region were substituted with A and T respectively to prevent binding of the CAP protein at low glucose concentrations. The wt -10 promoter sequence was also mutated from TATGTT to TATAAT in order to allow σ factor (RpoD) to bind without relying on further activation by the CAP protein. As a result of these mutations, gene expression mediated by the PL8-UV5 promoter will be independently regulated by intracellular concentrations of the lacI repressor as all CAP-associated regulation has been removed.

CviJI Endonuclease (+ ssrA deg. tag)

As part of the St Andrews iGEM 2020 kill switch mechanism, an enzyme capable of destroying plasmid inserts was of highest priority to prevent uptake of synthetic genes, whose expression by bacteria in the surrounding environment may have provided a survival advantage. All plasmid insert sequences were subject to commercial enzyme restriction site screening using the SnapGene feature which proposed the CviKI-1/CviJI restriction site to be most common across all sequences. In this case, the small size (4bp) of the restriction site is useful for our project.

The restriction endonuclease CviJI (also known as R.CviJI) is taken natively from the Chlorella virus IL-3A, a double-stranded DNA phycodnavirus that infects unicellular, eukaryotic Chlorella-like green algae. As well as being previously expressed in E.coli by Skowron et al. (1995) and Swaminathan et al. (1996), the restriction endonuclease is also used commercially and is available via NEB as CviKI-1. The enzyme cuts at RG/CY sites (where R = purines, Y = pyrimidines) in the presence of Mg2+. With the addition of ATP, R.CviJI (now R.CviJI*) cleaves at additional restriction sites RG/CN and YG/CY (where N = any nucleotide) but not YG/CR. Both enzymes cleave DNA frequently and therefore possess a variety of functions such as generating numerous sequence-specific oligonucleotides. The sequence to be used in this part is 278 amino acids in length and does not exhibit additional R.CviJI* activity. The 144-235 amino acid region is also suggested to have a recognition/catalytic domain.

R.CviJI will be fused to the ssrA degradation tag AANDENYADAS to prevent plasmid destruction as a result of leaky expression only. Lon protease, native to E.coli, will recognise and degrade the fusion construct. The TAG stop codon of the R.CviJI gene was removed and replaced with the AANDENYADAS sequence. TAATAA was then added to the 3' end of the the ssrA to terminate translation.


Design Notes

The wt sequence for the lac operon regulatory region was taken from Kalnins A. (1993) [GenBank accession no. J01636]. Within the CAP binding site, bases -66 (G) and -55 (C) of the wt binding region were substituted with A and T respectively to prevent binding of the CAP protein at low glucose concentrations. The wt -10 promoter sequence was also mutated from TATGTT to TATAAT in order to allow σ factor (RpoD) to bind without relying on further activation by the CAP protein (Reznikoff et al., 1978). This produced PL8-UV5.

As the cviJI gene is new to the registry, it must meet BBa assembly standards. A BsaI illegal restriction site was present at position 503. This was subsequently removed by in silico point mutagenesis: t501a (TCT (Ser) -> TCA (Ser)). As with other parts St. Andrews iGEM 2020 have characterised, a codon optimisation step was favourable however upon using the IDT codon optimisation tool, many additional CviJI restriction sites were introduced to the cviJI gene sequence. This was of course an issue as the gene coding for the enzyme would be fragmented, limiting the amount of expression of R.CviJI. As a result, codon optimisation was abandoned.

Additionally, a CviJI restriction site was already present in the initial gene sequence at position 477 (a perfect example of evolution at work given such a small frequency). To prevent the enzyme cutting its own gene sequence, the St Andrews iGEM team 2020 looked at in silico point mutagenesis for this site however unfortunately, no alternative was available as the codon involved in the restriction site was that of tryptophan (TGG/CCT). As this is the only CviJI restriction site present in the gene sequence, it is likely that this Trp residue is vital to the function of the enzyme. This was considered however to follow in the same HGT vein as with introducing R.CviJI in the first place as it meant a functioning endonuclease gene would less likely be transformed into other bacterial species.

As previously mentioned, placing a lon protease ssrA degradation tag onto R.CviJI would mop up any leaky expression of the fusion construct in order to prevent unwanted plasmid fragmentation too early. The strong AANDENYALAA degradation tag was initially suggested however it was thought this sequence was too strong to allow any endonuclease functionality when expressed. As a result, the tag was replaced with a 'moderately fast' AANDENYADAS degradation tag. The TAG stop codon of the R.CviJI gene was removed and replaced with the AANDENYADAS sequence. TAATAA was then added to the 3' end of the the ssrA to terminate translation.

The RBS BBa_J61100 was used as a very weak RBS (strength 4.29% relative to B0034 - Team Warsaw iGEM 2010) as strong expression of the endonuclease is not required for plasmid DNA fragmentation.


Source

The lac operon is found natively in E.coli. The part sequence was taken from BBa_K1695000 who generated the part sequence through data obtained from Reznikoff et al. (1978) and Hirschel et al. (1980). The sequence for R.CviJI, native to Chlorella virus IL-3A, was obtained from Skowron et al. (1995) and confirmed by Swaminathan et al. (1996) (ENA reference: U09001.1). The sequence for the ssrA degradation tag was obtained from registry part BBa_M0052 by the Endy lab (2007).

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