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Our Experimental Results

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Click elements of the diagram below to see results for each section of our project.











Looking for Interlab Study related results? Click below!

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 Results

Click the headings to see experiments and results

Sarcosine 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.

Rationale and Aim

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Background Information

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Design Stage

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Implementation

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Characterisation

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Conclusions and Future Work

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References

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Detector Modules: The Results

Click the headings to see experiments and results

Synthetic Promoter Library

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.

Rationale and Aim

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Background Information

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Design Stage

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Implementation

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Characterisation

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Conclusions and Future Work

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References

Arsenic Biosensor

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.

Rationale and Aim

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Background Information

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Design Stage

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Implementation

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Characterisation

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Conclusions and Future Work

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References

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Psicose Biosensor (Evry Paris-Saclay Collaboration)

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.

Rationale and Aim

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Background Information

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Design Stage

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Implementation

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Characterisation

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Conclusions and Future Work

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References

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Processor Modules: The Results

Click the headings to see experiments and results

Fim Standby Switch

BioBricks 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.

Rationale and Aim

Sensynova multicellular biosensor platform has been developed to overcome the limitations 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 part can be used within the platform as a Processor unit. Real world applications of biosensors are limited by many factors, one of which is that with most biosensors there is not a readout signal showing if the biosensor is working when not in use, i.e that the cells are still alive and have not lost their biosensor phenotypes. This can make them difficult to use, as well as market, since their viability comes into question as well as leading to false negatives/positives. Biosensors which rely on expression of a reporter signal may also suffer from unobserved activation due to weak or inconstant induction.

For this section of the project, as an improvement on a part by the Tokyo Tech team, we aim to produce a biobrick compatible part which is able to constitutively express a reporter signal prior to activation (to show that it is functioning) and to amplify a weak or inconsistent induction signal by permanently switching from an [OFF] to [ON] state after induction.

Background Information

Expression of the E. coli type 1 fimbriae gene is tightly regulated and phase dependent, i.e expression is either completely [ON] or [OFF] >>FimB FimE fimbrae control - Klemm 1986<<. This change in expression is controlled by the action of two proteins FimB and FimE which independently act upon a 300bp promoter region upstream of the fimbriae gene. The 300bp promoter region is inverted to either activate or suppress expression >> Roles of FimB FimE in site specific inversion - McClain 1991<<. Typical gene regulation mechanisms rely on up or down regulation of a promoter from a baseline expression, the fimbriae mechanism of ‘ALL’ or ‘NONE’ makes it a useful tool for synthetic biology applications. While the FimB protein inverts the promoter back and forth between [ON] and [OFF] states the FimE protein permanently inverts the promoter from [ON] to [OFF]. This inversion can be used to amplify weak or inconsistent induction signals.

The part we are producing is a combination of the 2015 Tokyo Tech part (BBa_K1632013, BBa_K1632007) and the Voight lab 2007 part (BBa_J64718) quorum sensing RhlI an autoinducer synthesis protein which produces C4-HSL. The part by Tokyo Tech used FimE in combination with GFP to switch from an [ON] to [OFF] state, we have produced a construct where in the [ON] state the non-fluorescense based reporter protein eforRed (Uppsala 2011 BBa_K592012) is expressed. In the [OFF] state the RhlI quorum sensing protein from P. aeruginosa is expressed. The eforRed chromoprotein was chosen as fluorescence based reporters often require complex laboratory equipment to measure expression.

Using a chromoprotein allows for the switch from [ON] to [OFF] to be verified by sight alone. The promoter flip also creates expression of the quorum sensing protein RhlI. This allows for a downstream activation of a reporter cell, meaning that the Fim Standby Switch has dual reporting properties.

The use of the FimE switch as opposed to FimB (which is reversible) means that even with a low or inconsistent induction signal, the permanent inversion of the 300bp fim promoter region leads to a signal which can be observed. This may have applications in agricultural biosensors where there can be plant uptake of the potential induction compound (reference ). FimE was also chosen because FimB can switch the promoter back and forth, so it is likely to result in about half the plasmids with the promoter in one direction and half in the other over time, resulting in no strong signal either direction.

The inactivation of the eforRed chromoprotein following an induction signal visibly displays the [ON] to [OFF] inversion. Expression of this chromoprotein also displays that the [ON] section of the fim switch is working correctly.

Design Stage

To construct the Fim reporter switch 3 separate gBlocks were designed with overlapping adapter regions homologous to the iGEM prefix and suffix to allow for Gibson assembly into the pSB1C3 backbone whilst retaining biobrick compatibility. The individual genes and other components are shown in (Table 1). The 1st gBlock sequence starts with a RBS (B0034) upstream of the fimE ORF (K137007) with no promoter region, this is to allow for other promoters to be cloned in upstream of the part. Downstream of the fimE gene is a double terminator (B0015). All RBS and terminator sequences used are B0034 and B0015 respectively.

Implementation

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Characterisation

The fim Standby Switch has two main functions; a visual signal to show that the target compound has been detected and AHL production so that the part can be detected by a reporter cell. To characterise the part these functions are individually tested, in aim to further isolate issues if they occur.
To test the detecting function of the Fim Standby Switch it was assembled with the PBAD/AraC promotor. The PBAD/AraC promotor with Standby Switch parts were plated out onto four different LB plates containing chloramphenicol, two plates with different concentrations of glucose and chloramphenicol, and another containing arabinose. As colonies for the Fim Switch section were red and white when plated onto chloramphenicol plates due to leakiness, the plates with glucose in theory should suppress this switching and a greater percentage colonies on these plates should be red after transformation. The colonies on the arabinose plate should be white as translation of fimE leads to the flipping fimS, and expression of RHlI.

The number of colonies on the plate that are white and red confirm inversion, this will show the percentage of colonies in the [ON] and [OFF] states. DNA sequencing will show inversion of the switch. As fimE is unidirectional over time the colonies should all become white on the plate containing arabinose. The plate containing glucose should repress leakage and the medium is supplemented by some percentage glucose.

To ensure that AHL is produced a white colony is chosen, where the fimS section has flipped, and a red colony which has not yet inverted. These colonies are then co-cultured with a successfully independently tested reporter cell. This reporter cell detects AHL production and as a result GFP produced. This also shows that the reversed sequences for RHLI and B0034 are working as expected.

Look at this fancy picture of our first part of Fim Standby Switch Characterisation to avoid the fact that we are still facing problems with it.

Conclusions and Future Work

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References

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Signal Tuners

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.

Rationale and Aim

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Background Information

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Design Stage

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Implementation

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Characterisation

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Conclusions and Future Work

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References

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Reporter Modules: The Results

Click the headings to see experiments and results

deGFP

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.

Rationale and Aim
Background Information
Design Stage
Implementation
Characterisation
Conclusions and Future Work
References

Chromoproteins

BioBricks 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.

Rationale and Aim

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).

Background Information

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.

BBa_K1033915 – amajLime

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.

BBa_K1033925 – spisPink

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.

BBa_K1033929 – aeBlue

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.

Design Stage

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.

Implementation
Characterisation
Conclusions and Future Work
References

Sensynova Framework Testing (IPTG Sensor): The Results

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.

Rationale and Aim

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Background Information

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Design Stage

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Implementation

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Characterisation

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Conclusions and Future Work

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References

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Cell Free Protein Synthesis System Optimisation: The Results

BioBricks used: BBa_K515105 (Imperial College London 2011)

Cell Free Protein Synthesis Premix Supplements: Diagrammatic overview of CFPS supplement roles in transcription and translation.

Rationale and Aim

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.

While CFPS systems are promising alternatives to whole cells, there are currently some drawbacks, primarily their cost and variability between batches of cell extract. To address these issues, this part of the project had the following aims: (i) develop a functional CFPS system, (ii) demonstrate the usefulness of a Design of Experiments (DoE) approach towards identifying which supplements are the most crucial for maximal CFPS activity, and (iii) demonstrate how DoE can be used to optimise each batch of cell extract for maximal CFPS activity.

Background Information

Cell Free Protein Synthesis Systems
Cell free protein synthesis (CFPS) systems are capable of performing transcription and translation of exogenous DNA in vitro. CFPS systems have been in use for many decades (Nirenberg & Matthaei, 1961), however the field of synthetic biology has resulted in a CFPS renaissance (Lu, 2017; Lee & Kim, 2013). Commonly, CFPS systems are based on cell extracts, which provide the transcription/translation machinery, as well as enzymes required to generate ATP required for protein synthesis.

While CFPS systems have a lot of potential, they also suffer from some drawbacks. Two of the major issues are the large variation in CFPS activity between cell extracts (Katsura, et al., 2017), and the high costs compared to whole cells (although cost have been reduced significantly in the past decade) (Carlson, et al., 2012). These issues can hinder the uptake of CFPS systems as an alternative chassis to whole cells, and as research tools.

Click to show/hide more information about the CFPS premix

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).

Design Stage
Implementation
Characterisation
Conclusions and Future Work
References

@@ Line 1,365: / Line 1,365: @@
 Expression of the E. coli type 1 fimbriae gene is tightly regulated and phase dependent, i.e expression is either completely [ON] or [OFF] >>FimB FimE fimbrae control - Klemm 1986<<.  This change in expression is controlled by the action of two proteins FimB and FimE which independently act upon a 300bp promoter region upstream of the fimbriae gene.  The 300bp promoter region is inverted to either activate or suppress expression >> Roles of FimB FimE in site specific inversion - McClain 1991<<.  Typical gene regulation mechanisms rely on up or down regulation of a promoter from a baseline expression, the fimbriae mechanism of ‘ALL’ or ‘NONE’ makes it a useful tool for synthetic biology applications.  While the FimB protein inverts the promoter back and forth between [ON] and [OFF] states the FimE protein permanently inverts the promoter from [ON] to [OFF].  This inversion can be used to amplify weak or inconsistent induction signals.
 <br /><br />
-The part we are producing is a combination of the 2015 Tokyo Tech part (BBa_K1632013, BBa_K1632007) and the Voight lab 2007 part (BBa_J64718) quorum sensing RhlI an autoinducer synthesis protein which produces C4-HSL. The part by Tokyo Tech used FimE in combination with GFP to switch from an [ON] to [OFF] state, we have produced a construct where in the [ON] state the non-fluorescense based reporter protein eforRed (Uppsala 2011 BBa_K592012) is expressed.  In the [OFF] state the RhlI quorum sensing protein from P. aeruginosa is expressed.  The eforRed chromoprotein was chosen as fluorescence based reporters often require complex laboratory equipment to measure expression.  Using a chromoprotein allows for the switch from [ON] to [OFF] to be verified by sight alone.  <br /><br />
+The part we are producing is a combination of the 2015 Tokyo Tech part (BBa_K1632013, BBa_K1632007) and the Voight lab 2007 part (BBa_J64718) quorum sensing RhlI an autoinducer synthesis protein which produces C4-HSL. The part by Tokyo Tech used FimE in combination with GFP to switch from an [ON] to [OFF] state, we have produced a construct where in the [ON] state the non-fluorescense based reporter protein eforRed (Uppsala 2011 BBa_K592012) is expressed.  In the [OFF] state the RhlI quorum sensing protein from P. aeruginosa is expressed.  The eforRed chromoprotein was chosen as fluorescence based reporters often require complex laboratory equipment to measure expression. <br /><br />
-The promoter flip also creates expression of the quorum sensing protein RhlI.  This allows for a downstream activation of a reporter cell, meaning that the Fim Standby Switch has dual reporting properties.
+Using a chromoprotein allows for the switch from [ON] to [OFF] to be verified by sight alone. The promoter flip also creates expression of the quorum sensing protein RhlI.  This allows for a downstream activation of a reporter cell, meaning that the Fim Standby Switch has dual reporting properties.
 <br /><br />
 The use of the FimE switch as opposed to FimB (which is reversible) means that even with a low or inconsistent induction signal, the permanent inversion of the 300bp fim promoter region leads to a signal which can be observed.  This may have applications in agricultural biosensors where there can be plant uptake of the potential induction compound (reference ).  FimE was also chosen because FimB can switch the promoter back and forth, so it is likely to result in about half the plasmids with the promoter in one direction and half in the other over time, resulting in no strong signal either direction.<br /><br />