Difference between revisions of "Team:TUDelft/Demonstrate"
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<li><a class="jump" href="#Overview">Overview</a></li> | <li><a class="jump" href="#Overview">Overview</a></li> | ||
<li><a class="jump" href="#CellFreeReplication">Cell-free Replication</a></li> | <li><a class="jump" href="#CellFreeReplication">Cell-free Replication</a></li> | ||
| − | <li><a class="jump" href="# | + | <li><a class="jump" href="#Transcription">Transcriptional Variance</a></li> |
<li><a class="jump" href="#CrossSpecies">Cross-species stabilization</a></li> | <li><a class="jump" href="#CrossSpecies">Cross-species stabilization</a></li> | ||
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| − | + | <p>Our team's mission was to set the basis for a universal mobile genetic tool which could be readily used to engineer a wide range of diverse bacterial species- contributing to a streamlined, host-independent multi-chassis Synthetic Biology. To achieve this, we set ourselves two main goals: the first goal was to investigate the implementation and viability of the phi29 bacteriophage linear DNA replication system as an orthogonal replication tool in bacterial cells. The second goal was to engineer a system which generates constant gene expression levels independently of transcriptional and translational variations that are expected to occur when transferring circuits across organisms.</p> | |
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<p>In this project, we have successfully demonstrated independence of expression levels from transcriptional variations with our novel incoherent feed-forward loop (IFFL) design, and have achieved cross-species expression level stabilization between <i>Escherichia coli</i> and <i>Pseudomonas putida</i>. Moreover, we have validated replication of our synthetic phi29 plasmid in cell-free systems as well as performed titration experiments to uncover the effects of different concentrations of phi29 replication-associated proteins in <i>E. coli</i >.</p> | <p>In this project, we have successfully demonstrated independence of expression levels from transcriptional variations with our novel incoherent feed-forward loop (IFFL) design, and have achieved cross-species expression level stabilization between <i>Escherichia coli</i> and <i>Pseudomonas putida</i>. Moreover, we have validated replication of our synthetic phi29 plasmid in cell-free systems as well as performed titration experiments to uncover the effects of different concentrations of phi29 replication-associated proteins in <i>E. coli</i >.</p> | ||
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<h1>Cell-free replication of linear DNA construct</h1> | <h1>Cell-free replication of linear DNA construct</h1> | ||
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| − | <p>We have <a href=https://2019.igem.org/Team:TUDelft/Design#control | + | <p>We have <a href="https://2019.igem.org/Team:TUDelft/Design#control">designed</a> and constructed an orthogonal transcription IFFL based with the use of T7 promoter variants.</p> |
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| − | <p>Based on <a href=https://2019.igem.org/Team:TUDelft/Model#TranscriptionalVariations | + | <p>Based on <a href="https://2019.igem.org/Team:TUDelft/Model#TranscriptionalVariations">kinetic model solutions</a>, we predicted that the system would be stable in GFP expression idenpendently of variations in trascription. Firstly, we demonstrated this control across varying T7 RNAP concentrations (caused by different IPTG titrations in the <i>E. coli</i> BL21 expression strain). |
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<figcaption class="centermodel"><b><br>Figure 1</b>: GFP fluorescence of IFFL optimized construct and unregulated GFP under T7sp1 promoter in <i>E. coli</i> BL21 DE3 at different IPTG concentrations. </figcaption> | <figcaption class="centermodel"><b><br>Figure 1</b>: GFP fluorescence of IFFL optimized construct and unregulated GFP under T7sp1 promoter in <i>E. coli</i> BL21 DE3 at different IPTG concentrations. </figcaption> | ||
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| − | <br><p>As it can be observed, both native strength and medium strength T7 promoter versions of our IFFL exhibited simmilar fluorescence. Furthermore, tunability was shown by the change in promoter strength ratios (weak promoter in TALEsp1 repressor).The data corroborates with the effective repression of our <a href = "https://2019.igem.org/Team:TUDelft/Design#moclo"novel TALE regulated T7 promoters. For more details <a href = "https://2019.igem.org/Team:TUDelft/Results#Transcription">click here</a>.</p><br><br> | + | <br><p>As it can be observed, both native strength and medium strength T7 promoter versions of our IFFL exhibited simmilar fluorescence. Furthermore, tunability was shown by the change in promoter strength ratios (weak promoter in TALEsp1 repressor).The data corroborates with the effective repression of our <a href = "https://2019.igem.org/Team:TUDelft/Design#moclo">novel TALE regulated T7 promoters. For more details <a href = "https://2019.igem.org/Team:TUDelft/Results#Transcription">click here</a>.</p><br><br> |
<h1>Cross-species gene expression stabilization</h1> | <h1>Cross-species gene expression stabilization</h1> | ||
Revision as of 20:22, 21 October 2019
Our team's mission was to set the basis for a universal mobile genetic tool which could be readily used to engineer a wide range of diverse bacterial species- contributing to a streamlined, host-independent multi-chassis Synthetic Biology. To achieve this, we set ourselves two main goals: the first goal was to investigate the implementation and viability of the phi29 bacteriophage linear DNA replication system as an orthogonal replication tool in bacterial cells. The second goal was to engineer a system which generates constant gene expression levels independently of transcriptional and translational variations that are expected to occur when transferring circuits across organisms.
In this project, we have successfully demonstrated independence of expression levels from transcriptional variations with our novel incoherent feed-forward loop (IFFL) design, and have achieved cross-species expression level stabilization between Escherichia coli and Pseudomonas putida. Moreover, we have validated replication of our synthetic phi29 plasmid in cell-free systems as well as performed titration experiments to uncover the effects of different concentrations of phi29 replication-associated proteins in E. coli.
Cell-free replication of linear DNA construct
Transcriptional variation
We have designed and constructed an orthogonal transcription IFFL based with the use of T7 promoter variants.
Figure 1: Design of our orthogonal transcription iFFL for the stabilization of expression across organisms. T7 promoter variants ensure constant ratio of transcription in varying T7 RNAP concentrations and activity. The use of the same RBS in both repressor and output gene result constant ratio of translation initiation in both genes. This results in independence of gene expression levels from variations in these rates.
Based on kinetic model solutions, we predicted that the system would be stable in GFP expression idenpendently of variations in trascription. Firstly, we demonstrated this control across varying T7 RNAP concentrations (caused by different IPTG titrations in the E. coli BL21 expression strain).
Figure 1: GFP fluorescence of IFFL optimized construct and unregulated GFP under T7sp1 promoter in E. coli BL21 DE3 at different IPTG concentrations.
When compared to uncontrolled GFP, the IFFL clearly exhibited steady expression in the range of 0 to 1 mM IPTG concentrations.
Robustness to strength of promoter variations was also demonstrated by comparing IFFL circuits with the same T7 promoter variant in each gene.
As it can be observed, both native strength and medium strength T7 promoter versions of our IFFL exhibited simmilar fluorescence. Furthermore, tunability was shown by the change in promoter strength ratios (weak promoter in TALEsp1 repressor).The data corroborates with the effective repression of our novel TALE regulated T7 promoters. For more details click here.
Cross-species gene expression stabilization
We have succeeded in cloning a broad host range promoter version of our IFFL in Pseudomonas putida for comparison with E. coli, as well as negative control constitutive promoter.
Figure 2: Design of our broad host range transcription iFFL for the stabilization of expression across organisms. The system was tested in E. coli and P. putida.
We observed a reduction in both absolute and relative differences of gene expression levels. Although these constructs are influenced by a multiple variables, we believe, however that this is a right step in the direction of stabilization across organisms, although further calibration of the system for higher expression would be useful. Also, we have successfully engineered a constitutive promoter which functions effectively in both E. coli and P. putida.
