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− | As part of our project, SOX was designed to be an ‘adapter’ that could link glyphosate into our framework via a formaldehyde detector module. This concept could then be applied to other molecules that have easily detectable substrates in their degradation pathways. The aim of this part of the project was to demonstrate that SOX can be expressed by E. coli cells and that when glyphosate is added SOX can convert it to formaldehyde to be detected via a biosensor. | + | As part of our project, SOX was designed to be an ‘adapter’ that could link glyphosate into our framework via a formaldehyde detector module. This concept could then be applied to other molecules that have easily detectable substrates in their degradation pathways. The aim of this part of the project was to demonstrate that SOX can be expressed by <i>E. coli</i> cells and that when glyphosate is added SOX can convert it to formaldehyde to be detected via a biosensor. |
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<p class="SOX"> | <p class="SOX"> | ||
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− | To ensure the codon usage of our SOX protein was not differing significantly from the average codon usage of E. coli, rare codons were removed from the sequence using the <a href="https://www.idtdna.com/CodonOpt">IDT codon optimisation tool</a>to produce high protein expression. | + | To ensure the codon usage of our SOX protein was not differing significantly from the average codon usage of <i>E. coli</i>, rare codons were removed from the sequence using the <a href="https://www.idtdna.com/CodonOpt">IDT codon optimisation tool</a>to produce high protein expression. |
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− | E. coli BL21-DE3 cells have higher levels of protein expression than DH5α cells and so were a more practical choice. This led to the expression of SOX being placed under the control of a T7 promoter due to BL21-DE3 cells producing T7 polymerase after the addition of IPTG. | + | <i>E. coli</i> BL21-DE3 cells have higher levels of protein expression than DH5α cells and so were a more practical choice. This led to the expression of SOX being placed under the control of a T7 promoter due to BL21-DE3 cells producing T7 polymerase after the addition of IPTG. |
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<p class="PSI">The plasmid backbone was acquired by digestion [Protocol link] of the part K2205015 with XbaI and SpeI, cutting out the original sfGFP construct. </p> | <p class="PSI">The plasmid backbone was acquired by digestion [Protocol link] of the part K2205015 with XbaI and SpeI, cutting out the original sfGFP construct. </p> | ||
− | <p class="PSI">The Psicose detector construct was assembled into the plasmid backbone using the NEB Hi-Fi kit [Protocol link] and transformed into DH5α E. coli cells [Protocol link]. </p> | + | <p class="PSI">The Psicose detector construct was assembled into the plasmid backbone using the NEB Hi-Fi kit [Protocol link] and transformed into DH5α <i>E. coli</i> cells [Protocol link]. </p> |
<p class="PSI"> Colonies picked from streaked plates and cultures were prepared for miniprepping [Protocol link]. DNA samples were then sent off for sequencing [Website link] to ensure that the constructs were correct. </p> | <p class="PSI"> Colonies picked from streaked plates and cultures were prepared for miniprepping [Protocol link]. DNA samples were then sent off for sequencing [Website link] to ensure that the constructs were correct. </p> | ||
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<p class=" SIG "> The part K2205010 contained in pSB1C3, was digested [Protocol link] using SpeI and PstI to allow for the insertion of the processing variants directly after the Las controlled promoter (pLas) that would trigger transcription of sensitivity tuners in the presence of connector 1 of the Sensynova platform. </p> | <p class=" SIG "> The part K2205010 contained in pSB1C3, was digested [Protocol link] using SpeI and PstI to allow for the insertion of the processing variants directly after the Las controlled promoter (pLas) that would trigger transcription of sensitivity tuners in the presence of connector 1 of the Sensynova platform. </p> | ||
− | <p class=" SIG "> Ligations were set up overnight [Protocol link] using NEB’s T4 ligase and transformed in DH5α E. coli cells [Protocol link]. Colony PCR [Protocol link] was performed to check ligations. Colonies picked for this protocol were streaked onto a LB-agar plate. </p> | + | <p class=" SIG "> Ligations were set up overnight [Protocol link] using NEB’s T4 ligase and transformed in DH5α <i>E. coli</i> cells [Protocol link]. Colony PCR [Protocol link] was performed to check ligations. Colonies picked for this protocol were streaked onto a LB-agar plate. </p> |
<p class=" SIG "> Colonies picked from streaked plates and cultures were prepared for miniprepping [Protocol link]. Minipreps were digested [Protocol link] with SpeI and PstI to allow for the insertion of the part K2205011 directly after the PO promoter. </p> | <p class=" SIG "> Colonies picked from streaked plates and cultures were prepared for miniprepping [Protocol link]. Minipreps were digested [Protocol link] with SpeI and PstI to allow for the insertion of the part K2205011 directly after the PO promoter. </p> | ||
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<p class=" SIG "> The part K2205010 contained in pSB1C3, was digested [Protocol link] using XbaI and PstI for BioBrick assembly [Protocol link]. | <p class=" SIG "> The part K2205010 contained in pSB1C3, was digested [Protocol link] using XbaI and PstI for BioBrick assembly [Protocol link]. | ||
− | Ligations were set up overnight [Protocol link] using NEB’s T4 ligase and transformed in DH5α E. coli cells [Protocol link]. Colony PCR [Protocol link] was performed to check ligations. Colonies picked for this protocol were streaked onto a LB-agar plate. </p> | + | Ligations were set up overnight [Protocol link] using NEB’s T4 ligase and transformed in DH5α <i>E. coli</i> cells [Protocol link]. Colony PCR [Protocol link] was performed to check ligations. Colonies picked for this protocol were streaked onto a LB-agar plate. </p> |
<p class=" SIG "> Colonies picked from streaked plates and cultures were prepared for miniprepping [Protocol link]. DNA samples were then sent off for sequencing [Website link] to ensure that the constructs were correct. </p> | <p class=" SIG "> Colonies picked from streaked plates and cultures were prepared for miniprepping [Protocol link]. DNA samples were then sent off for sequencing [Website link] to ensure that the constructs were correct. </p> | ||
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<!--- How was this part of the project implemented? How was it assembled if it is a part? How was it prepared for testing? What were the challenges? Why was it implemented in that way (e.g. why was that assembly method chosen)? How was it confirmed as having been implemented/assembled correctly? Include gels, images of plates, sequence data, preliminary information, etc. and refer to the specific sections of the lab-book which document this. ---> | <!--- How was this part of the project implemented? How was it assembled if it is a part? How was it prepared for testing? What were the challenges? Why was it implemented in that way (e.g. why was that assembly method chosen)? How was it confirmed as having been implemented/assembled correctly? Include gels, images of plates, sequence data, preliminary information, etc. and refer to the specific sections of the lab-book which document this. ---> | ||
− | <p class="CHR"> The chromoproteins aeBlue (BBa_K1033929), amajLime (BBa_K1033915) and spisPink (BBa_K1033925) parts were requested from the iGEM parts registry. Upon arrival, parts were transformed in DH5α E. coli cells [Protocol link]. Colonies were picked and overnight cultures were prepared for miniprepping [Protocol link]. Minipreps were digested [Protocol link] with XbaI and PstI for BioBrick assembly [Protocol link]. </p> | + | <p class="CHR"> The chromoproteins aeBlue (BBa_K1033929), amajLime (BBa_K1033915) and spisPink (BBa_K1033925) parts were requested from the iGEM parts registry. Upon arrival, parts were transformed in DH5α <i>E. coli</i> cells [Protocol link]. Colonies were picked and overnight cultures were prepared for miniprepping [Protocol link]. Minipreps were digested [Protocol link] with XbaI and PstI for BioBrick assembly [Protocol link]. </p> |
<p class="CHR">The part K2205013 contained in pSB1C3, was digested [Protocol link] using SpeI and PstI to allow for the insertion of the chromoproteins directly after the RhI controlled promoter (pRhI) that would trigger transcription of colour proteins in the presence of connector 2 of the Sensynova platform. </p> | <p class="CHR">The part K2205013 contained in pSB1C3, was digested [Protocol link] using SpeI and PstI to allow for the insertion of the chromoproteins directly after the RhI controlled promoter (pRhI) that would trigger transcription of colour proteins in the presence of connector 2 of the Sensynova platform. </p> | ||
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Energy can also be derived from glutamate in the supplement solution (Jewett, et al., 2008), which is added in the form of magnesium glutamate and potassium glutamate. Glutamate is a metabolite in the tricarboxylic acid cycle, which generates NADH. In whole cells, NADH is used in oxidative phosphorylation to produce ATP. Oxidative phosphorylation relies on membrane bound proteins and proton gradients across a membrane. It has been shown previously that extracts prepared using French Press or sonication contain membrane vesicles which have ATPase activity (Futai, 1974), and that oxidative phosphorylation can be activated in CFPS systems (Jewett, et al., 2008). | Energy can also be derived from glutamate in the supplement solution (Jewett, et al., 2008), which is added in the form of magnesium glutamate and potassium glutamate. Glutamate is a metabolite in the tricarboxylic acid cycle, which generates NADH. In whole cells, NADH is used in oxidative phosphorylation to produce ATP. Oxidative phosphorylation relies on membrane bound proteins and proton gradients across a membrane. It has been shown previously that extracts prepared using French Press or sonication contain membrane vesicles which have ATPase activity (Futai, 1974), and that oxidative phosphorylation can be activated in CFPS systems (Jewett, et al., 2008). | ||
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− | Sodium oxalate, another component of the supplement solution, is also used to help increase energy generation by the system. PEP synthetase, an enzyme present in E. coli, converts pyruvate into phosphoenol pyruvate (PEP) in a reaction which consumes ATP, thereby wasting ATP and directing it away from protein synthesis. Oxalate inhibits PEP synthetase by acting as a pyruvate mimic, and hence limit the energy wasted by this reaction. | + | Sodium oxalate, another component of the supplement solution, is also used to help increase energy generation by the system. PEP synthetase, an enzyme present in <i>E. coli</i>, converts pyruvate into phosphoenol pyruvate (PEP) in a reaction which consumes ATP, thereby wasting ATP and directing it away from protein synthesis. Oxalate inhibits PEP synthetase by acting as a pyruvate mimic, and hence limit the energy wasted by this reaction. |
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− | The ribonucleotides ATP, GTP, UTP, and CTP are also components of the supplement solution. They are used in the synthesis of mRNA for transcription of desired genes encoding on exogenous DNA added to the system, and ATP can also be used directly as energy for translation. The polyamines spermidine and putrescine are two other supplements which are added to aid with transcription. It is thought that they can bind proteins and DNA to help recruit polymerase for transcription. Polyamines may also increase translation fidelity, aid ribosome assembly, and activate tRNAs (Jelenc & Kurland, 1979; Jewett & Swartz, 2004b; Algranati & Goldemberg, 1977). To enable translation to occur, amino acids (added separately from the supplement solution) and an E. coli tRNA mixture are added to the CFPS system. Folinic acid is also added as it can be used as a source of folinate for the synthesis of f-Met; the amino acid required for initiation of translation in E. coli. | + | The ribonucleotides ATP, GTP, UTP, and CTP are also components of the supplement solution. They are used in the synthesis of mRNA for transcription of desired genes encoding on exogenous DNA added to the system, and ATP can also be used directly as energy for translation. The polyamines spermidine and putrescine are two other supplements which are added to aid with transcription. It is thought that they can bind proteins and DNA to help recruit polymerase for transcription. Polyamines may also increase translation fidelity, aid ribosome assembly, and activate tRNAs (Jelenc & Kurland, 1979; Jewett & Swartz, 2004b; Algranati & Goldemberg, 1977). To enable translation to occur, amino acids (added separately from the supplement solution) and an <i>E. coli</i> tRNA mixture are added to the CFPS system. Folinic acid is also added as it can be used as a source of folinate for the synthesis of f-Met; the amino acid required for initiation of translation in <i>E. coli</i>. |
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Magnesium and potassium ions are also added as supplements. Both ions are ubiquitous in cells with many functions in protein synthesis, namely aiding translation by associating with ribosome subunits and stabilising RNA (Nierhaus, 2014; Pyle, 2002). While magnesium ions are essential for protein synthesis, at high concentrations they can cause inhibition of ribosome translocation and hence inhibit protein synthesis (Borg & Ehrenberg, 2015). | Magnesium and potassium ions are also added as supplements. Both ions are ubiquitous in cells with many functions in protein synthesis, namely aiding translation by associating with ribosome subunits and stabilising RNA (Nierhaus, 2014; Pyle, 2002). While magnesium ions are essential for protein synthesis, at high concentrations they can cause inhibition of ribosome translocation and hence inhibit protein synthesis (Borg & Ehrenberg, 2015). | ||
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<p> | <p> | ||
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− | Cell free extract preparation procedures were based on methods reported in literature previously (Kwon & Jewett, 2015). Cell free extracts were prepared from <i>Escherichia coli</i> BL21 and <i>Bacillus subtilis</i> 168. Cells were streak plated out from glycerol stocks on LB agar (15 mg/mL agar, 10 mg/mL tryptone, 5 mg/mL yeast extract, 0.17 M sodium chloride) and incubated overnight at 37<sup>o</sup>C. A single colony was used to inoculate 10 mL LB broth (10 mg mL<sup>-1</sup> tryptone, 5 mg mL<sup>-1</sup> yeast extract, 0.17 M sodium chloride) before shake-incubation at 37<sup>o</sup>C for approximately 16 hours overnight. 2 mL of overnight liquid culture was used to inoculate 200 mL LB broth in a 2 L flask and shake-incubated at 37<sup>o</sup>C until late exponential phase was reached (OD<sub>600 nm</sub> of approximately 2.5 for E. coli BL21 cells). The culture was split in half and cells were harvested by centrifugation at 4,500 RPM and 4<sup>o</sup>C for 20 minutes in pre-weighed falcon tubes. The wet cell pellet weight was determined before storage at -20<sup>o</sup>C. Cells were defrosted on ice for approximately 1.5 hours and resuspended in approximately 10 mL of ice-cold CFPS wash buffer (60 mM potassium glutamate, 14 mM magnesium glutamate, 10 mM TRIS (pH 8.2 with acetic acid); autoclave sterilised; supplemented with 2 mM DTT immediately before use) per gram of wet cell pellet. Resuspended cells were centrifuged at 4,500 RPM and 4oC for 20 mins. The supernatant was discarded and cell pellets were resuspended and centrifuged in CFPS wash buffer twice more. The washed pellets were then resuspended in 1 mL CFPS wash buffer per gram of wet cell pellet and aliquoted to 1 mL in 2 mL tubes. Cells were lysed by sonication (20% amplitude, cycles of 40 seconds on – 59.9 seconds off, 432.5 Joules) and the lysates were clarified by centrifugation at 12,000 RPM for 10 mins, flash frozen in liquid nitrogen, and stored at -80oC. | + | Cell free extract preparation procedures were based on methods reported in literature previously (Kwon & Jewett, 2015). Cell free extracts were prepared from <i>Escherichia coli</i> BL21 and <i>Bacillus subtilis</i> 168. Cells were streak plated out from glycerol stocks on LB agar (15 mg/mL agar, 10 mg/mL tryptone, 5 mg/mL yeast extract, 0.17 M sodium chloride) and incubated overnight at 37<sup>o</sup>C. A single colony was used to inoculate 10 mL LB broth (10 mg mL<sup>-1</sup> tryptone, 5 mg mL<sup>-1</sup> yeast extract, 0.17 M sodium chloride) before shake-incubation at 37<sup>o</sup>C for approximately 16 hours overnight. 2 mL of overnight liquid culture was used to inoculate 200 mL LB broth in a 2 L flask and shake-incubated at 37<sup>o</sup>C until late exponential phase was reached (OD<sub>600 nm</sub> of approximately 2.5 for <i>E. coli</i> BL21 cells). The culture was split in half and cells were harvested by centrifugation at 4,500 RPM and 4<sup>o</sup>C for 20 minutes in pre-weighed falcon tubes. The wet cell pellet weight was determined before storage at -20<sup>o</sup>C. Cells were defrosted on ice for approximately 1.5 hours and resuspended in approximately 10 mL of ice-cold CFPS wash buffer (60 mM potassium glutamate, 14 mM magnesium glutamate, 10 mM TRIS (pH 8.2 with acetic acid); autoclave sterilised; supplemented with 2 mM DTT immediately before use) per gram of wet cell pellet. Resuspended cells were centrifuged at 4,500 RPM and 4oC for 20 mins. The supernatant was discarded and cell pellets were resuspended and centrifuged in CFPS wash buffer twice more. The washed pellets were then resuspended in 1 mL CFPS wash buffer per gram of wet cell pellet and aliquoted to 1 mL in 2 mL tubes. Cells were lysed by sonication (20% amplitude, cycles of 40 seconds on – 59.9 seconds off, 432.5 Joules) and the lysates were clarified by centrifugation at 12,000 RPM for 10 mins, flash frozen in liquid nitrogen, and stored at -80oC. |
A CFPS supplement solution was prepared based on previously reported protocols (Yang, et al., 2012). Briefly, amino acid stock solutions were prepared according to table 1. Briefly, amino acids were weighed in 2 mL tubes, dissolved in 5 M potassium hydroxide, and stored at -20oC. A 10x amino acid solution was prepared by mixing the stock solutions together in amounts according to Appendix.2 and the pH was adjusted to 7.9 with acetic acid. The solution was aliquoted to 1.5 mL and stored at -80oC. | A CFPS supplement solution was prepared based on previously reported protocols (Yang, et al., 2012). Briefly, amino acid stock solutions were prepared according to table 1. Briefly, amino acids were weighed in 2 mL tubes, dissolved in 5 M potassium hydroxide, and stored at -20oC. A 10x amino acid solution was prepared by mixing the stock solutions together in amounts according to Appendix.2 and the pH was adjusted to 7.9 with acetic acid. The solution was aliquoted to 1.5 mL and stored at -80oC. | ||
− | The following solutions were prepared in autoclave sterilised MiliQ water and stored at -80oC: 100x magnesium glutamate solution (1.2 M magnesium glutamate), 10x salt solution (1.3 M potassium glutamate, 40 mM sodium oxalate, 10 mM ammonium acetate), 25x NTPS & co-factor mix (37.5 mM spermidine, 30 mM ATP, 21.25 mM GTP, UTP, and CTP, 25 mM putrescine, 8.25 mM nicotinamide diphosphate, 4.25 mg mL-1 E. coli tRNA (Roche), 0.85 mg mL-1 folinic acid, N xX co-enzyme A), 25x sodium pyruvate solution (825 mM sodium pyruvate, pH to 7.3 with potassium hydroxide), unless stated otherwise. A 5x CFPS supplement solution premix (5% v/v nuclease free water, 5% v/v magnesium glutamate solution, 50% v/v salt solution, 20% v/v NTPS & co-factor mix, 20% v/v sodium pyruvate solution, unless stated otherwise) was prepared and stored at -80oC. | + | The following solutions were prepared in autoclave sterilised MiliQ water and stored at -80oC: 100x magnesium glutamate solution (1.2 M magnesium glutamate), 10x salt solution (1.3 M potassium glutamate, 40 mM sodium oxalate, 10 mM ammonium acetate), 25x NTPS & co-factor mix (37.5 mM spermidine, 30 mM ATP, 21.25 mM GTP, UTP, and CTP, 25 mM putrescine, 8.25 mM nicotinamide diphosphate, 4.25 mg mL-1 <i>E. coli</i> tRNA (Roche), 0.85 mg mL-1 folinic acid, N xX co-enzyme A), 25x sodium pyruvate solution (825 mM sodium pyruvate, pH to 7.3 with potassium hydroxide), unless stated otherwise. A 5x CFPS supplement solution premix (5% v/v nuclease free water, 5% v/v magnesium glutamate solution, 50% v/v salt solution, 20% v/v NTPS & co-factor mix, 20% v/v sodium pyruvate solution, unless stated otherwise) was prepared and stored at -80oC. |
CFPS activity of systems prepared as above were tested by expression of 1.7 μg pSB1C3-J23100-sfGFP (Figure CFPS-1). Firstly, enough CFPS master mix was prepared for 7 reactions by mixing 112 μL cell extract, 70 μL CFPS supplement premix, and 21 μL amino acid solution in a 1.5 mL tube and stored on ice. A further six 1.5 mL tubes were put on ice; 21 μL of nuclease free water was added to three tubes, and 1.7 μg pSB1C3-J23100-sfGFP plasmid DNA from the same stock solution was added to the remaining three. Tubes containing DNA were made up to 21 μL with nuclease-free water. CFPS master mix (29 μL) was then added to all tubes, which were vortexed and transferred to a 96-well plate. The plate was incubated in a BMG Labtech Fluostar Optima at 370C for 4.25 hours with fluorescence readings (excitation: 485 nm, emission: 510 nm) every 15 mins. Figure CFPS-2 shows that over time, fluorescence intensity increased in systems with DNA encoding for sfGFP compared to systems with no DNA. Hence, the system had CFPS activity. | CFPS activity of systems prepared as above were tested by expression of 1.7 μg pSB1C3-J23100-sfGFP (Figure CFPS-1). Firstly, enough CFPS master mix was prepared for 7 reactions by mixing 112 μL cell extract, 70 μL CFPS supplement premix, and 21 μL amino acid solution in a 1.5 mL tube and stored on ice. A further six 1.5 mL tubes were put on ice; 21 μL of nuclease free water was added to three tubes, and 1.7 μg pSB1C3-J23100-sfGFP plasmid DNA from the same stock solution was added to the remaining three. Tubes containing DNA were made up to 21 μL with nuclease-free water. CFPS master mix (29 μL) was then added to all tubes, which were vortexed and transferred to a 96-well plate. The plate was incubated in a BMG Labtech Fluostar Optima at 370C for 4.25 hours with fluorescence readings (excitation: 485 nm, emission: 510 nm) every 15 mins. Figure CFPS-2 shows that over time, fluorescence intensity increased in systems with DNA encoding for sfGFP compared to systems with no DNA. Hence, the system had CFPS activity. | ||
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<div class="result_img"><img class="result_img" src="https://static.igem.org/mediawiki/2017/e/e3/T--Newcastle--BB_CFPS_initial_test.png" width="100%"/> | <div class="result_img"><img class="result_img" src="https://static.igem.org/mediawiki/2017/e/e3/T--Newcastle--BB_CFPS_initial_test.png" width="100%"/> | ||
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− | <p class="legend"><strong>Figure CFPS-2 Initial Testing of CFPS Systems:</strong> Negative corrected fluorescence for E. coli BL21 extract-based CFPS systems. Each data point is an average of 3 replicate reactions, and error bars represent +/- standard error.</p> | + | <p class="legend"><strong>Figure CFPS-2 Initial Testing of CFPS Systems:</strong> Negative corrected fluorescence for <i>E. coli</i> BL21 extract-based CFPS systems. Each data point is an average of 3 replicate reactions, and error bars represent +/- standard error.</p> |
</div> | </div> | ||
</td> | </td> |
Revision as of 15:48, 25 October 2017
Our Experimental ResultsClick elements of the diagram below to see results for each section of our project. Alternatively, click here to see a list of our experiments and results. Want to learn more about our framework (above)? Head over to our description page! |
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Biochemcial Adaptor Modules: The ResultsSarcosine Oxidase (Glyphosate to Formaldehyde)BioBricks used: BBa_0123456 (New), BBa_7890123 (Team_Name 20XX)
Diagrammatic Overview: This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. This is a caption.
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Detector Modules: The ResultsSynthetic Promoter LibraryBioBricks used: BBa_0123456 (New), BBa_7890123 (Team_Name 20XX)
Diagrammatic Overview: This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. This is a caption.
Arsenic BiosensorBioBricks used: BBa_0123456 (New), BBa_7890123 (Team_Name 20XX)
Diagrammatic Overview: This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. The Sensynova multicellular biosensor platform has been developed to overcome the limitations identified by our team [hyperlink to human practices] that hamper the success in biosensors development. One of these limits regards the lack of modularity and reusability of the various components. Our platform design, based on the expression of three main modules (Detector, Processor and Output) by three E.coli strains in co-culture, allows the switch of possible variances for each module and the production of multiple customised biosensors. This section of the project is based on testing the modularity of the system by replacing the IPTG detector part of the Sensynova design with different detecting parts. In particular, an Arsenic sensing part will be used.
Psicose Biosensor (Evry Paris-Saclay Collaboration)BioBricks used: BBa_K2205023 (New), BBa_??? (Evry Paris-Saclay 2017)
Diagrammatic Overview: This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. The Sensynova multicellular biosensor platform has been developed to overcome the limitations identified by our team [hyperlink to human practices] that hamper the success in biosensors development. One of these limits regards the lack of modularity and reusability of the various components. Our platform design, based on the expression of three main modules (Detector, Processor and Output) by three E.coli strains in co-culture, allows the switch of possible variances for each module and the production of multiple customised biosensors. This section of the project is based on testing the modularity of the system by implementing the biosensor created by the 2017 Evry Paris-Saclay iGEM team into the Sensynova platform as part of our collaboration requirement. This biosensor was designed, made and submitted to the iGEM registry by the Evry Paris-Saclay 2017 team. We chose to use this system as a variant to the IPTG detector module present in the Sensynova platform in order to fulfil the requirement of collaborating with another iGEM team. The image below, provided to us by the Evry Paris-Saclay 2017 team, details the psicose biosensor design. It features the pLac derivative promoter pTAC (BBa_K180000), a RBS (BBa_B0034), the PsiR coding sequence, the terminator (BBa_B0015), the synthetic promoter pPsitac, a RBS (BBa_B0034), a mCherry coding sequence and finally the terminator (BBa_B0015) flanked by the iGEM prefix and suffix. The inducible system works as detailed in the diagram below. When pTAC is induced due to the presence of IPTG, PsiR is transcribed and binds to the pPsitac promoter repressing the transcription of the mCherry protein. When psicose is present, the sugar binds to PsiR, freeing up the promoter and subsequently the colour output. In order to implement the psicose biosensor variant to the Sensynova platform, a design was created by replacing the IPTG sensing system in the original detector module with the construct detailed above, creating part K2205023. We chose to replace the pTAC promoter with the constitutive promoter present within the platform in order to eliminate the need for induction with IPTG. In place of the colour output present in the Evry Paris-Saclay design, we have added our part K2205008, which produces our first connector in order to trigger a response from following modules of the Sensynova platform. Part K2205023 detailed above was designed using Benchling and ordered for synthesis through IDT. Using Benchling, virtual digestions and ligations were simulated resulting in the plasmid map detailed below. The Psicose detector construct obtained by gBlock synthesis has been designed to include required overhangs for Gibson assembly into the linearized plasmid pSB1C3. The plasmid backbone was acquired by digestion [Protocol link] of the part K2205015 with XbaI and SpeI, cutting out the original sfGFP construct. The Psicose detector construct was assembled into the plasmid backbone using the NEB Hi-Fi kit [Protocol link] and transformed into DH5α E. coli cells [Protocol link]. Colonies picked from streaked plates and cultures were prepared for miniprepping [Protocol link]. DNA samples were then sent off for sequencing [Website link] to ensure that the constructs were correct.
Due to time constraints resulted from synthesis delays, we lacked the time to co-culture this part with the Sensynova platform's multiple modules in order for the creation of variants. The part K2205023, the Evry Pasir-Sclay's psicose biosensor system as the detecting unit of the platform, has been submitted to the iGEM registry for future work and characterisation by future teams. |
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Processor Modules: The ResultsFim Standby SwitchBioBricks used: BBa_0123456 (New), BBa_K1632013, BBa_K1632007(2015 Tokyo Tech part)
Figure X: The Fim Switch in the native [OFF] state where the eforRED reporter is expressed allowing direct visualisation of the cells.
Signal TunersBioBricks used: BBa_K2205024 (New),BBa_K2205025 (New), BBa_K274371 (Cambridge 2009), BBa_K274381 (Cambridge 2009)
Diagrammatic Overview: This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. The Sensynova multicellular biosensor platform has been developed to overcome the limitations identified by our team [hyperlink to human practices] that hamper the success in biosensors development. One of these limits regards the lack of modularity and reusability of the various components. Our platform design, based on the expression of three main modules (Detector, Processor and Output) by three E.coli strains in co-culture, allows the switch of possible variances for each module and the production of multiple customised biosensors. This section of the project is based on testing the modularity of the system by inserting two different sensitivity tuner constructs between the processing units of the Sensynova platform; BBa_K274371 and BBa_K274381. Both selected sensitivity tuner constructs were made and submitted to the iGEM registry by the Cambridge 2009 team. They were chosen as variants to the empty processing module present in the Sensynova platform due to the fact that, although they have been included in the iGEM distribution kit since their submission in 2009, they have yet to be successfully implemented into a team’s system, as far as we are aware. The 2007 Cambridge iGEM team built 15 different constructs that amplified the PoPS output of the promoter pBad/AraC detailed by image below taken from the Cambridge 2009 team's wiki. FIGURE LEGEND The 2009 Cambridge iGEM team then re-designed these constructs to be PoPS converters, as image below taken from their wiki details, and generated a set sensitivity tuners corresponding to Cambridge 2007’s amplifiers. This part is made up of a RBS (BBa_B0034), an org activator coding sequence (BBa_I746350) from P2 phage, the double terminator BBa_B0015 (made up of BBa_B0010 and BBa_B0012) and the inducible promoter PO (BBa_I746361) from P2 phage. This part is made up of a RBS (BBa_B0034), a pag activator coding sequence (BBa_I746351) from PSP3 phage, the double terminator BBa_B0015 (made up of BBa_B0010 and BBa_B0012) and the inducible promoter PO (BBa_I746361) from P2 phage. In order to implement these two sensitivity tuner variants into the Sensynova platform, designs were made by inserting the above parts between the two constructs forming the empty processor module of our framework. Using Benchling, virtual digestions of the two sensitivity tuners and ligations to the part K2205010, the connector 1 receiver module, were carried out. These two new constructs were then virtual digested and ligated to the part K2205011, the connector 2 reporter module, resulting in the two plasmid maps detailed below; parts K2205024 and K2205025. The sensitivity tuners parts BBa_K274371 and BBa_K274381 were requested from the iGEM parts registry. Upon arrival, parts were transformed in DH5α E. coli cells [Protocol link]. Colonies were picked and cultures were prepared for miniprepping [Protocol link]. Minipreps were digested [Protocol link] with XbaI and PstI for BioBrick assembly [Protocol link]. The part K2205010 contained in pSB1C3, was digested [Protocol link] using SpeI and PstI to allow for the insertion of the processing variants directly after the Las controlled promoter (pLas) that would trigger transcription of sensitivity tuners in the presence of connector 1 of the Sensynova platform. Ligations were set up overnight [Protocol link] using NEB’s T4 ligase and transformed in DH5α E. coli cells [Protocol link]. Colony PCR [Protocol link] was performed to check ligations. Colonies picked for this protocol were streaked onto a LB-agar plate. Colonies picked from streaked plates and cultures were prepared for miniprepping [Protocol link]. Minipreps were digested [Protocol link] with SpeI and PstI to allow for the insertion of the part K2205011 directly after the PO promoter. The part K2205010 contained in pSB1C3, was digested [Protocol link] using XbaI and PstI for BioBrick assembly [Protocol link]. Ligations were set up overnight [Protocol link] using NEB’s T4 ligase and transformed in DH5α E. coli cells [Protocol link]. Colony PCR [Protocol link] was performed to check ligations. Colonies picked for this protocol were streaked onto a LB-agar plate. Colonies picked from streaked plates and cultures were prepared for miniprepping [Protocol link]. DNA samples were then sent off for sequencing [Website link] to ensure that the constructs were correct. Due to time constraints, we lacked the time to characterise these parts into the Sensynova platform within the lab. The parts K2205024 and K2205025, the parts BBa_K274371 and BBa_K274381 respectively as processing units of the platform, were been submitted to the iGEM registry for future work and characterisation by future teams. | |||
Reporter Modules: The ResultsdeGFPBioBricks used: BBa_0123456 (New), BBa_7890123 (Team_Name 20XX)
Diagrammatic Overview: This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. ChromoproteinsBioBricks used: BBa_K2205016 (New),BBa_K2205017 (New),BBa_K2205018 (New), BBa_K1033915 (Uppsala 2013), BBa_K1033925 (Uppsala 2013), BBa_K1033929 (Uppsala 2013)
Diagrammatic Overview: This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. The Sensynova multicellular biosensor platform has been developed to overcome the limitations identified by our team [hyperlink to human practices] that hamper the success in biosensors development. One of these limits regards the lack of modularity and reusability of the various components. Our platform design, based on the expression of three main modules (Detector, Processor and Output) by three E.coli strains in co-culture, allows the switch of possible variances for each module and the production of multiple customised biosensors. This section of the project is based on testing the modularity of the system by replacing the sfGFP output part of the Sensynova platform design with three different output chromoprotein variants; BBa_K1033929 (aeBlue), BBa_K1033925 (spisPink) and BBa_K1033915 (amajLime). All three selected chromoproteins were made and submitted to the iGEM registry by the Uppsala 2013 team. They were chosen as variants to the sfGFP present in the Sensynova platform as they exhibit of strong colour readily observed in both LB cultures and in agar plates when expressed. All three proteins have significant sequence homologies with proteins in the GFP family. The amajLime protein is a yellow-green chromoprotein extracted from the coral Anemonia majano. It was first extracted and characterized by Matz et al. under the name amFP486 (UniProtKB/Swiss-Prot: Q9U6Y6.1 GI: 56749103 GenBank: AF168421.1) and codon optimized for E coli by Genscript. The protein has an absorption maximum at 458 nm giving it a yellow-green colour visible to the naked eye. The spisPink protein is a pink chromoprotein extracted from the coral Stylophora pistillata. It was first extracted and characterized by Alieva et al. under the name spisCP (GenBank: ABB17971.1) and codon optimized for E coli by Genscript. The protein has an absorption maximum at 560 nm giving it a pink colour visible to the naked eye. The strong colour is readily observed in both LB or on agar plates after less than 24 hours of incubation. The aeBlue protein is a blue chromoprotein extracted from the basal disk of a beadlet anemone Actinia equine. It was first extracted and characterized by Shkrob et al. 2005 under the name aeCP597 and codon optimised for E coli by Bioneer Corp. The protein has an absorption maximum at 597nm and a deep blue colour visible to the naked eye. The protein aeBlue has significant sequence homologies with proteins in the GFP family. The coding sequence for this protein was originally submitted to the registry as BBa_K1033916 by the 2012 Uppsala iGEM team. In order to implement these three chromoprotein variants into the Sensynova platform, designs were made by replacing the sfGFP in the original reporter module with the parts detailed above that were ordered from the iGEM parts registry. Using Benchling, virtual digestions of the three chromoproteins and ligations to the part K2205013, the connector 2 receiver module detailed above, were carried out resulting in the three plasmid maps detailed below; parts K2205016, K2205017 and K220518. The chromoproteins aeBlue (BBa_K1033929), amajLime (BBa_K1033915) and spisPink (BBa_K1033925) parts were requested from the iGEM parts registry. Upon arrival, parts were transformed in DH5α E. coli cells [Protocol link]. Colonies were picked and overnight cultures were prepared for miniprepping [Protocol link]. Minipreps were digested [Protocol link] with XbaI and PstI for BioBrick assembly [Protocol link]. The part K2205013 contained in pSB1C3, was digested [Protocol link] using SpeI and PstI to allow for the insertion of the chromoproteins directly after the RhI controlled promoter (pRhI) that would trigger transcription of colour proteins in the presence of connector 2 of the Sensynova platform. Stared colonies picked from streaked plates and cultures were prepared for miniprepping [Protocol link]. DNA samples were then sent off for sequencing [Website link] to ensure that the constructs were correct. Alieva, N., Konzen, K., Field, S., Meleshkevitch, E., Hunt, M., Beltran-Ramirez, V., Miller, D., Wiedenmann, J., Salih, A. and Matz, M. (2008). Diversity and Evolution of Coral Fluorescent Proteins. PLoS ONE, 3(7), p.e2680. Matz, M., Fradkov, A., Labas, Y., Savitsky, A., Zaraisky, A., Markelov, M. and Lukyanov, S. (1999). Nature Biotechnology, 17(10), pp.969-973. Shkrob, M., Yanushevich, Y., Chudakov, D., Gurskaya, N., Labas, Y., Poponov, S., Mudrik, N., Lukyanov, S. and Lukyanov, K. (2005). Far-red fluorescent proteins evolved from a blue chromoprotein fromActinia equina. Biochemical Journal, 392(3), pp.649-654. |
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Sensynova Framework Testing (IPTG Sensor): The ResultsBioBricks used: BBa_0123456 (New), BBa_7890123 (Team_Name 20XX)
Diagrammatic Overview: This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. This is a caption.
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Cell Free Protein Synthesis System Optimisation: The ResultsBioBricks used: BBa_K515105 (Imperial College London 2011)
Cell Free Protein Synthesis Premix Supplements: Diagrammatic overview of CFPS supplement roles in transcription and translation.
Cell free protein synthesis (CFPS) systems have large potential as alternative chassis for applications such biosensors or diagnostic tests. This is because generally, biosensors are needed to function outside of the laboratory environment. Whole cells, which are traditionally used as chassis, can be problematic in these scenarios due to issues with containment and release of genetically modified organisms.
Cells extracts being used in CFPS systems tend to be supplemented with a cocktail of compounds and molecules to aid the process of transcription and translation. Although exact supplement solutions can vary from protocol to protocol, most have the same basic composition; salts, nucleotides, tRNAs, co-factors, energy sources, and amino acids (Yang, et al., 2012). The supplement solution used in this study is based on the Cytomin system (figure 1.2.1) (Jewett, et al., 2008). For the cytomin supplement solution, the major energy source is sodium pyruvate, which is converted to acetate through a series of reactions catalysed by enzymes in the crude cell extract (Figure 1.2.2). The first reaction, pyruvate to acetyl-CoA, requires nicotinamide diphosphate (NAD) and Co-enzyme A (CoA) as co-factors. Both of these are components of the premix and hence added to the system to enhance flux through the reaction. The acetyl CoA is phosphorylated by inorganic phosphate, and then de-phosphorylated to produce ATP from ADP. The ATP is used as energy to drive translation of mRNA. Energy can also be derived from glutamate in the supplement solution (Jewett, et al., 2008), which is added in the form of magnesium glutamate and potassium glutamate. Glutamate is a metabolite in the tricarboxylic acid cycle, which generates NADH. In whole cells, NADH is used in oxidative phosphorylation to produce ATP. Oxidative phosphorylation relies on membrane bound proteins and proton gradients across a membrane. It has been shown previously that extracts prepared using French Press or sonication contain membrane vesicles which have ATPase activity (Futai, 1974), and that oxidative phosphorylation can be activated in CFPS systems (Jewett, et al., 2008). Sodium oxalate, another component of the supplement solution, is also used to help increase energy generation by the system. PEP synthetase, an enzyme present in E. coli, converts pyruvate into phosphoenol pyruvate (PEP) in a reaction which consumes ATP, thereby wasting ATP and directing it away from protein synthesis. Oxalate inhibits PEP synthetase by acting as a pyruvate mimic, and hence limit the energy wasted by this reaction. The ribonucleotides ATP, GTP, UTP, and CTP are also components of the supplement solution. They are used in the synthesis of mRNA for transcription of desired genes encoding on exogenous DNA added to the system, and ATP can also be used directly as energy for translation. The polyamines spermidine and putrescine are two other supplements which are added to aid with transcription. It is thought that they can bind proteins and DNA to help recruit polymerase for transcription. Polyamines may also increase translation fidelity, aid ribosome assembly, and activate tRNAs (Jelenc & Kurland, 1979; Jewett & Swartz, 2004b; Algranati & Goldemberg, 1977). To enable translation to occur, amino acids (added separately from the supplement solution) and an E. coli tRNA mixture are added to the CFPS system. Folinic acid is also added as it can be used as a source of folinate for the synthesis of f-Met; the amino acid required for initiation of translation in E. coli. Magnesium and potassium ions are also added as supplements. Both ions are ubiquitous in cells with many functions in protein synthesis, namely aiding translation by associating with ribosome subunits and stabilising RNA (Nierhaus, 2014; Pyle, 2002). While magnesium ions are essential for protein synthesis, at high concentrations they can cause inhibition of ribosome translocation and hence inhibit protein synthesis (Borg & Ehrenberg, 2015).
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