Team:Newcastle/Results
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! |
||||||||||||||||||||||||||||||||||||||||||||||
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.
|
||||||||||||||||||||||||||||||||||||||||||||||
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.
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. [Insert image of modules here] 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 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 constitutively expressed, 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. 
|
||||||||||||||||||||||||||||||||||||||||||||||
Processor Modules: The ResultsFim Standby SwitchBioBricks used: BBa_0123456 (New), BBa_7890123 (Team_Name 20XX)
Diagrammatic Overview: Representation of the switching mechanism of the Fim Switch, in the native [OFF] state the eforRED reporter is expressed (shown in red) allowing direct visualisation of the cells. Following the inversion of the promoter region (K1632004), eforRED expression is halted and the RhlI gene is expressed (J64718), this is now the [ON] state.
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. [Insert image of modules here] 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. 
FIGURE LEGEND The 2009 Cambridge iGEM team then re-designed these constructs to be PoPS converters, as image below 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.
| ||||||||||||||||||||||||||||||||||||||||||||||
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. [Insert image of modules here] 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. |
||||||||||||||||||||||||||||||||||||||||||||||
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.
|
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). | |||||||||||||||||||||||||||||||||||||||||||||
