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                <h2>Overview</h2>
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                <p>The behavior of genetic parts and circuits in different bacterial species is unpredictable as it is influenced by host-dependent variations. We identified the variables to be: copy number, transcriptional and translational rates. We implemented a unique control system motif (incoherent feed forward loop) into a genetic circuit to achieve gene expression independent of copy number, transcriptional and translational rates.</p>  
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                        <a class="toggle " href="javascript:void(0);" ><b>Design</b><span style="float:right;"><b>&#xfe40;</b></span></a>
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                            <p>We modeled the genetic implementation of the iFFL loop and varied the identified variables. Based on the results from the modeling, we made design choices.
  
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                                <h2>Results</h2>  
  
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                                We learned through the implementation of the model that constant transcriptional and translational rates of TALE and GFP needs to be maintained to achieve gene expression independent of transcriptional and translational variations respectively.
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<h1>Plasmid Copy Number </h1>
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                                <img src="https://static.igem.org/mediawiki/2019/f/f8/T--TUDelft--transcriptionvariation.svg" style="width:85%;border:1px solid #00a6d6;" class="centermodel"
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                                    alt="TALE system">
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<h1>Transcriptional Variations</h1>
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                                <figcaption class="centermodel"><b>Figure 5</b>: Steady-state GFP production while transcription rates of both TALE and GOI are changed (aT/aG = constant). The lines indicate constant ratio of transcription rates </figcaption>
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                                To achieve constant ratios of transcriptional rates of TALE and GFP, we used the orthogonal T7 promoter and its variants to express TALE and GFP genes. The following constructs were successfully cloned by Golden Gate Assembly. 
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                                    To achieve constant ratios of translational rates for TALE and GFP, we used the same ribosome binding sites for the expression of TALE and GFP. Furthermore, to demonstrate expression independent of translational rates, we switched constructed circuits with different RBSs.
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<h1>Translational Variantions</h1>
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                                        When transcriptional units are placed in series, leaky expression of the gene in the second transcriptional unit can occur. This is due to the efficiency of the terminator of the first transcriptional unit. The model shows that leaky expression significantly affects the ability of the iFFL system to adapt to changes in copy number.
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<h1>Harmonization (qualitative)</h1>
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                                        <img src="https://static.igem.org/mediawiki/2019/0/0e/T--TUDelft--leakyterminator.svg" style="width:70%;border:1px solid #00a6d6;" class="centermodel"
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                                        <figcaption class="centermodel"><b>Figure 9</b>: Comparison of a perfect terminator and a leaky terminator on the expression level at different plasmid copy number. </figcaption>
  
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                                        We therefore designed our genetic circuit such that the transcriptional units of TALE and GFP are oriented in opposite directions.
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            <div id="CopyNumber">
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                <h1>Plasmid Copy Number </h1>
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                <p>text</p>
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            </div>
  
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                <h3>References</h3>
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                <h1>Transcriptional Variations</h1>
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                <p>text</p>
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                            <a target="_blank" href="http://doi.org/10.3389/fmicb.2018.02154">Sun, D. (2018). Pull in and Push Out: Mechanisms of Horizontal Gene Transfer in Bacteria. Frontiers in Microbiology, 9, 2154. https://doi.org/10.3389/fmicb.2018.02154</a>
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<li><a target="_blank" id="Weber2011" href="http://doi.org/10.1371/journal.pone.0016765">Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PloS One, 6(2), e16765. http://doi.org/10.1371/journal.pone.0016765</a></li>
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                <h1>Harmonization (qualitative)</h1>
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                <p>text</p>
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            <br>
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                <h1>Future Plan </h1>
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                <p>text</p>
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            <h3>References</h3>
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            <div class="reftu">
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                <ul style="list-style:none;">
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                    <li>
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                        <a target="_blank" href="http://doi.org/10.3389/fmicb.2018.02154">Sun, D. (2018). Pull in and Push Out: Mechanisms of Horizontal Gene Transfer in Bacteria. Frontiers in Microbiology, 9, 2154. https://doi.org/10.3389/fmicb.2018.02154</a>
 +
                    </li>
 +
                    <li><a target="_blank" id="Weber2011" href="http://doi.org/10.1371/journal.pone.0016765">Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PloS One, 6(2), e16765. http://doi.org/10.1371/journal.pone.0016765</a></li>
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Revision as of 21:25, 20 October 2019

Sci-Phi 29

Overview

Parts Construction

text


Part Characterization

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Orthogonalibity

text


Orthogonal Replication

text

Toxicity Assay

text


Controllabillity

Overview

The behavior of genetic parts and circuits in different bacterial species is unpredictable as it is influenced by host-dependent variations. We identified the variables to be: copy number, transcriptional and translational rates. We implemented a unique control system motif (incoherent feed forward loop) into a genetic circuit to achieve gene expression independent of copy number, transcriptional and translational rates.

  • Design﹀

      We modeled the genetic implementation of the iFFL loop and varied the identified variables. Based on the results from the modeling, we made design choices.

      Results

      We learned through the implementation of the model that constant transcriptional and translational rates of TALE and GFP needs to be maintained to achieve gene expression independent of transcriptional and translational variations respectively. TALE system
      Figure 5: Steady-state GFP production while transcription rates of both TALE and GOI are changed (aT/aG = constant). The lines indicate constant ratio of transcription rates
      To achieve constant ratios of transcriptional rates of TALE and GFP, we used the orthogonal T7 promoter and its variants to express TALE and GFP genes. The following constructs were successfully cloned by Golden Gate Assembly. To achieve constant ratios of translational rates for TALE and GFP, we used the same ribosome binding sites for the expression of TALE and GFP. Furthermore, to demonstrate expression independent of translational rates, we switched constructed circuits with different RBSs. When transcriptional units are placed in series, leaky expression of the gene in the second transcriptional unit can occur. This is due to the efficiency of the terminator of the first transcriptional unit. The model shows that leaky expression significantly affects the ability of the iFFL system to adapt to changes in copy number. TALE system
      Figure 9: Comparison of a perfect terminator and a leaky terminator on the expression level at different plasmid copy number.
      We therefore designed our genetic circuit such that the transcriptional units of TALE and GFP are oriented in opposite directions.


Plasmid Copy Number

text


Transcriptional Variations

text


Translational Variantions

text

Harmonization (qualitative)

text


Future Plan

text



References