Difference between revisions of "Part:BBa K2918040"

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<partinfo>BBa_K2918040 short</partinfo>
 
<partinfo>BBa_K2918040 short</partinfo>
  
Genetic implementation of an incoherent feed-forward loop (iFFL) in which a stabilized 0.1 T7 promoter is controlling GFP expression.
+
Genetic implementation of an incoherent feed-forward loop (iFFL) in which a weak strength T7 promoter with a binding site for TALE is controlling GFP expression.
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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===Usage and Biology===
 
===Usage and Biology===
An Incoherent feed-forward loop (iFFL) is a unique control systems motif where the output signal is robust to changes in the input signal. This is achieved by the introduction of a repressor.  
+
An incoherent feed-forward loop (iFFL) is a unique control systems motif where the output signal is robust to changes in the input signal (as shown in Figure 1). This is achieved by the introduction of a repressor.  
  
 
<div><ul>  
 
<div><ul>  
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</center>
 
</center>
 
     </ul></div>
 
     </ul></div>
iFFL can be applied to genetic circuits to achieve expression independent from copy number, transcriptional and translational rates. To implement the iFFL in a genetic circuit, <html><a href="https://parts.igem.org/Part:BBa_K2918008/">TALE </a></html> proteins can be used. These proteins consist of repeats where 12th and 13th amino acids can vary, these are called the repeat variable diresidue (RVD). RVDs have been shown to bind to DNA in a simple one-to-one binding code <html><a href="#Doyle2013">(Doyle, Stoddard et al., 2013)</a></html>. The direct correspondence between amino acids allows scientists to engineer these repeat regions to target any sequence they want. In our system, we used the TALE protein as a repressor by engineering promoters to contain the binding site of this specific TALE protein (<html><a href="https://parts.igem.org/Part:BBa_K2918009/">0.1 T7sp1 promoter</a></html>, <html><a href="https://parts.igem.org/Part:BBa_K2918010">0.5 T7sp1 promoter</a></html> and <html><a href="https://parts.igem.org/Part:BBa_K2918011">P<sub>BHR</sub>sp1 promoter</a></html>). <br> In our genetic circuit, a unrepressed promoter controls the expression of TALE protein while the promoters with the TALE binding sites drive expression of GFP.  
+
iFFL can be applied to genetic circuits to achieve expression independent from copy number, transcriptional and translational rates. To implement the iFFL in a genetic circuit, TALE proteins can be used. These proteins consist of repeats where 12th and 13th amino acids can vary, these are called the repeat variable diresidue (RVD). RVDs have been shown to bind to DNA in a simple one-to-one binding code <html><a href="#Doyle2013">(Doyle, Stoddard et al., 2013)</a></html>. The direct correspondence between amino acids allows scientists to engineer these repeat regions to target any sequence they want. In our system, we used the TALE protein as a repressor by engineering promoters to contain the binding site of this specific TALE protein (<html><a href="https://parts.igem.org/Part:BBa_K2918010">T7sp1 promoter</a></html> and <html><a href="https://parts.igem.org/Part:BBa_K2918011">P<sub>BHR</sub>sp1 promoter</a></html>). <br> In our genetic circuit, a unrepressed promoter controls the expression of TALE protein while the promoters with the TALE binding sites drive expression of GFP. <br><br>
 +
 
 
<div><ul>  
 
<div><ul>  
 
<center>
 
<center>
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     </ul></div>
 
     </ul></div>
  
When transcriptional units are placed in series due to low effieciency of terminators, leaky expression of the gene in the neighbouring transcriptonal unit can occur. This significantly influences the behavior of the iFFL genetic circuit <html><a href="#Segall2018">(Segall-Shapiro et al., 2018)</a></html>. Hence, the transcriptional units in the circuit are oriented outward to achieve insulation from influence of the neighboring transcriptional unit.  
+
When transcriptional units are placed in series due to low effieciency of terminators, leaky expression of the gene in the neighboring transcriptonal unit can occur. This significantly influences the behavior of the iFFL genetic circuit as predicted by our <html><a target="_blank" href="https://2019.igem.org/Team:TUDelft/Model">model</a></html>. Hence, the transcriptional unit of TALE is oriented in a different orientation than the transcriptional unit of GFP (as shown in Figure 2).  
  
An interesting application of the iFFL is to achieve controllable gene expression across different bacterial species. Gene expression in different bacterial contexts is influenced by changes in copy number, transcriptional and translational rates. To achieve expression robust to changes in transcriptional and tranlational rates, the ratio of transcriptional and translational rates of GFP and repressor need to be constant. This can be achieved by using orthogonal T7 promoter and its variants (<html><a href="https://parts.igem.org/Part:BBa_K2918053">T7 promoter</a></html>, <html><a href="https://parts.igem.org/Part:BBa_K2918005">0.5 T7 promoter</a></html>,<html><a href="https://parts.igem.org/Part:BBa_K2918006">0.1 T7 promoter</a></html>, <html><a href="https://parts.igem.org/Part:BBa_K2918009">0.5 T7sp1 promoter</a></html> and <html><a href="https://parts.igem.org/Part:BBa_K2918010">T7sp1 promoter</a></html> ).   
+
An interesting application of the iFFL is to achieve controllable gene expression across different bacterial species. Gene expression in different bacterial contexts is influenced by changes in copy number, transcriptional and translational rates. To achieve expression robust to changes in transcriptional and tranlational rates, the ratio of transcriptional and translational rates of GFP and repressor need to be constant. This can be achieved by using orthogonal T7 promoter and its variants (<html><a href="https://parts.igem.org/Part:BBa_K2918053">T7 promoter</a></html>, <html><a href="https://parts.igem.org/Part:BBa_K2918005">Weak T7 promoter</a></html>, <html><a href="https://parts.igem.org/Part:BBa_K2918006">Medium T7 promoter</a></html>, <html><a href="https://parts.igem.org/Part:BBa_K2918009">Medium T7sp1 promoter</a></html> and <html><a href="https://parts.igem.org/Part:BBa_K2918010">T7sp1 promoter</a></html>).   
  
Apart from being able to achieve stable gene expression across different bacterial species, it is necessary to attain  different levels of gene expression. The T7 promoter based optimized iFFL can be used to obtain higher levels of gene of interest (GFP) expression as the expression of TALE is driven by a lower strength promoter (0.1 T7 promoter) compared to the promoter (0.5 T7sp1 promoter) driving GFP expression.
+
Apart from being able to achieve stable gene expression across different bacterial species, it is necessary to attain  different levels of gene expression. The T7 promoter based optimized iFFL can be used to obtain higher levels of gene of interest (GFP) expression as the expression of TALE is driven by a lower strength promoter (Weak T7 promoter) compared to the promoter (Medium T7sp1 promoter) driving GFP expression.
  
 
===Strain Construction===
 
===Strain Construction===
The construct was assembled by golden gate assembly based modular cloning system. First, the individual transcriptional units were cloned into level 1 destination vectors <html><a href="http://www.addgene.org/47761/" target="_blank">pICH47761</a></html> and <html><a href="http://www.addgene.org/47822/" target="_blank">pICH47822</a></html> by BpiI based golden gate assembly. The multi-transcriptional unit construct was assembled by a BsaI based golden gate. The assembly was a one-pot restriction-ligation reaction where the individual level 1 constructs were added along with the destination vector <html><a href="http://www.addgene.org/48055/" target="_blank">pICH48055</a></html>. The correct clone was distinguished by blue-white screening and the construct was confirmed by sequencing. The cloning protocol can be found in the MoClo section below.
+
The construct was assembled by golden gate assembly based modular cloning system. First, the individual transcriptional units were cloned into level 1 destination vectors <html><a href="http://www.addgene.org/47761/" target="_blank">pICH47761</a></html> and <html><a href="http://www.addgene.org/47822/" target="_blank">pICH47822</a></html> by BsaI based golden gate assembly. The multi-transcriptional unit construct was assembled by a BpiI based golden gate. The assembly was a one-pot restriction-ligation reaction where the individual level 1 constructs were added along with the destination vector <html><a href="http://www.addgene.org/48055/" target="_blank">pICH48055</a></html>. The correct clone was distinguished by blue-white screening and the construct was confirmed by sequencing. The cloning protocol can be found in the MoClo section below.
  
 
===Modular Cloning===
 
===Modular Cloning===
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<b>Note: The basic parts sequences of the Sci-Phi 29 collection in the registry contain only the part sequence and therefore contain no overhangs or restriction sites. For synthesizing MoClo compatible parts, refer to table 2. The complete sequence of our parts including backbone can be found <html><a href="http://2019.igem.org/Team:TUDelft/Experiments" target="_blank">here</a>.</html></b>
+
<b>Note: The basic parts sequences of the Sci-Phi 29 collection in the registry contain only the part sequence and therefore contain no overhangs or restriction sites. For synthesizing MoClo compatible parts, refer to table 2.</b>
  
  
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         </table>
 
         </table>
 
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</body>
 
+
    </body>
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</html>
 
</html>
  
 
===Characterization===
 
===Characterization===
  <p> We have characterized the behavior of this system under the induction of different concentrations of IPTG. According to our <html><body><a href="https://2019.igem.org/Team:TUDelft/Model"> model </a></body></html> this system always yields the same gene of interest (GOI) expression levels when the transcription rate of both genes (TALE and GOI) is changed in the same ratio as can be seen in figure 3. </p><br>
+
<p>  
<br>
+
According to our <html><a target="_blank" href="https://2019.igem.org/Team:TUDelft/Model">model</a></html>, the genetic implementation of the iFFL system yields the same gene of interest (GOI) expression levels when the transcriptional rate of both genes (TALE and GOI) are changed in the same ratio as can be seen in Figure 3. </p>
 +
<div><ul>  
 +
<center>
 +
  <li style="display: inline-block;"> [[File:T--TUDelft--transcriptionvariation1.jpg|thumb|none|550px|<b>Figure 3</b>: Steady-state GFP production while transcriptional rates of both TALE and GOI are changed. The lines indicate constant ratio of transcriptional rates]] </li>
 +
</center>
 +
    </ul></div>  
  
<html><body>    <img src="https://2019.igem.org/wiki/images/f/f8/T--TUDelft--transcriptionvariation.svg" style="width:85%;border:1px solid #00a6d6;" class="centermodel"
+
<p>For the system to be versatile, it is important to achieve different levels of output gene expression (GFP). Hence, in the optimized iFFL system the TALE repressor is expressed by a higher strength promoter compared to the promoter controlling GFP expression. </p>
      alt="TALE system">
+
    <figcaption class="centermodel"><b>Figure 3</b>: Steady-state GFP production while transcription rates of both TALE and GOI are changed. The lines indicate constant ratio of transcription rates </figcaption></body></html>
+
  
<br>
+
<p> To validate the insulation of the gene expression (GFP) in the iFFL system to changes in transcriptional rates, GFP fluorescence was measured at different IPTG concentrations by flow cytometry at log-phase of growth. Change in IPTG concentrations, changes <I>in vivo</I> concentrations of T7 RNAP and this contributes to variations in transcriptional rates. In unrepressed systems, the expression of the GOI is a function of IPTG concentrations. However, in iFFL systems due to the expression of TALE repressor, similar GFP expression across different IPTG concentrations is expected. </p>
  
<p>In order to measure steady-state GFP levels we measured fluorescence in log-phase using flow cytometry. </p>
+
<p> The iFFL system was validated by measuring GFP using flow cytometry. As a control, GFP under the control of T7sp1 promoter was used. As a reference for background fluorescence, <i>E. coli</i> BL21(DE3) cells without plasmids were used. The protocol for sample preparation can be found <html><body><a href="https://2019.igem.org/Team:TUDelft/Experiments">here</a></body></html>.</p>
  
The protocol for preparation of samples for the flow cytometry assay is as follows:
+
<p> Scatter plots (Figures 7 and 8) for the unprepressed control and T7 based optimized iFFL were gated by eye to select for the most dense regions. Figures 4 and 5 show fluorescence measurements for the gated populations of the unrepressed control and optimized iFFL system at different IPTG concentrations respectively. </p>  
<html>
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<body>
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<ol>
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<li>Samples were grown overnight</li>
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<li>Overnight cultures were diluted to OD = 0.01 into 1 mL, and grow for 2 hours on 37 degrees 250 rpm shaking in 2 mL Eppendorf tubes. </li>
+
<li>Overnight cultures were diluted 1:100 into 5 mL, and grow for 4 hours on 37 degrees 250 rpm shaking in 50 mL eppendorf tubes. Induce with IPTG. </li>
+
<li>Samples were kept at 4 degrees for 1 hour </li>
+
</ol>
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</body>
+
</html>
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<br>
+
  
In the measurement, <i>E. coli BL21</i> cells without a plasmid were used as a reference for background fluorescence. As a control, <html><body><a href="https://parts.igem.org/Part:BBa_K2918037"> harmonized eGFP </a></body></html> driven by the same promoter and RBS was used. The gating for flow cytometry was determined by eye by selecting the densest region of  <i>E. coli TOP10</i>. Furthermore, the fluorescence histogram was gated to discern between cells that were 'on' and 'off', as in expressing fluorescence or not. Only cells of similar forward and side scatter were compared.  The median fluorescence intensity of the blank is subtracted from the fluorescence intensity of the samples to correct for autofluorescence. In figure 6 we plot the corrected fluorescence of the samples. Figures 7 and 8 show the gating and the fluorescence histogram of each sample for the negative control. Figures 9 and 10 show the same but for our optimized stabilized iFFL systems. </p>
+
<div><ul>  
 +
<center>
 +
  <li style="display: inline-block;"> [[File:T--TUDelft--L1etafluorescence.png|thumb|none|550px|<b>Figure 4</b>: Fluorescence measurements from gated population of unrepressed control (for scatter plot refer Figure 7) at different IPTG concentrations. Black: <i>E. coli</I> BL21(DE3)(Green: 0mM, Red: 0.1mM, Blue: 0.5mM and Purple: 1mM ]] </li>
 +
</center>
 +
    </ul></div>  
  
<html><body><img src="https://2019.igem.org/wiki/images/a/a9/T--TUDelft--IPTGtitration.svg" style="width:60%;border:1px solid #00a6d6;" class="centermodel"
+
<div><ul>  
        alt="TALE system">
+
<center>
          <figcaption class="centermodel"><b>Figure 6</b>: Steady-state GFP fluorescence measurement of promoter variation using FACS. The graph depicts T7 and 0.5 T7 iFFL systems, expected to give the same fluorescence according to the model. As a control, GFP under control of an unrepressed T7 promoter was used. </figcaption>
+
  <li style="display: inline-block;"> [[File:T--TUDelft--LM23fluorescence.png|thumb|none|550px|<b>Figure 5</b>: Fluorescence measurements from gated population of optimized iFFL system (for scatter plot refer Figure 8) at different IPTG concentrations. Black: <i>E. coli</I> BL21(DE3), Green: 0mM, Red:0.1mM, Blue: 0.5mM and Purple: 1mM ]] </li>
</body></html>
+
</center>
        <br>
+
    </ul></div>  
<br>
+
<p> In Figure 4, it is seen that GFP fluorescence of the unrepressed control changes with IPTG concentrations. However,(in Figure 5) for the optimized iFFL system, the GFP fluorescence curves for all the IPTG concentrations overlap and therefore is independent of IPTG concentrations. </p>  
  
<p>Figure 6 shows that our optimized TALE system results in the same expression level independent of IPTG concentration, while in the unrepressed T7 system the expression increases with increased IPTG induction. <.p>
+
<div><ul>
<br> <br>
+
<center>
 +
  <li style="display: inline-block;"> [[File:T--TUDelft--IPTGtitration.png|thumb|none|550px|<b>Figure 6</b>: Steady state GFP fluorescnece measurements using flow cytometry at different IPTG concentrations. Measurements were made at log-phase of growth and the median fluorescence of the gated populations is plotted. ]] </li>
 +
</center>  
 +
</ul></div>
  
<html><body><img src = "https://static.igem.org/mediawiki/parts/8/8a/T--TUDelft--L1etascatter.png" alt="Modeling" style="width:60%";></body></html>
+
<p> From Figure 6, it is seen that despite changes in IPTG the GFP fluorescence remains the same while the GFP fluorescence of the control increases with IPTG concentrations. This suggests that the gene of interest expression is independent of in-vivo T7 RNAP concentrations (IPTG concentrations) thereby independent of changes in transcriptional rate. </p>
<html><body><figcaption><br><b>Figure 7: Scatter plot of forward and side scatter of <i>E. coli BL21</i> cells without a plasmid. The region selected is the gating we considered to obtain the values depicted in figure 6. </b></figcaption></body></html>
+
<br>
+
  
<html><body><img src = "https://static.igem.org/mediawiki/parts/b/b2/T--TUDelft--L1etafluorescence.png" alt="Modeling" style="width:60%";></body></html>
+
===Conclusion===
<html><body><figcaption><br><b>Figure 8: Raw fluorescence values of our negative control. Black is<i>E. coli BL21</i>cells without a plasmid. Green is 0 mM IPTG, blue is 0.1 mM, pink is 0.5 and purple is 1 mM IPTG induction. </b></figcaption></body></html>
+
<p> The characterization of the T7 based optimized iFFL system suggests that the circuit is robust to changes in transcriptional rates. </p>
  
<br>
 
  
<html><body><img src = "https://static.igem.org/mediawiki/parts/8/8f/T--TUDelft--LM23scatter.png" alt="Modeling" style="width:60%";></body></html>
+
===Raw Data===
<html><body><figcaption><br><b>Figure 9: Scatter plot of forward and side scatter of <i>E. coli BL21</i> cells without a plasmid. The region selected is the gating we considered to obtain the values depicted in figure 6. </b></figcaption></body></html>
+
<div><ul>  
<br>
+
<center>
 +
  <li style="display: inline-block;"> [[File:T--TUDelft--L1etascatter.png|thumb|none|550px|<b>Figure 8</b>: Scatter plot for unrepressed control (forward scatter vs side scatter)]] </li>
 +
</center>
 +
    </ul></div>  
  
<html><body><img src = "https://static.igem.org/mediawiki/parts/b/bc/T--TUDelft--LM23fluorescence.png" alt="Modeling" style="width:60%";></body></html>
+
<div><ul>  
<html><body><figcaption><br><b>Figure 10: Raw fluorescence values of our stabilized system. Black is<i>E. coli BL21</i>cells without a plasmid. Green is 0 mM IPTG, blue is 0.1 mM, pink is 0.5 and purple is 1 mM IPTG induction. </b></figcaption></body></html>
+
<center>
<br>
+
  <li style="display: inline-block;"> [[File:T--TUDelft--LM23scatter.png|thumb|none|550px|<b>Figure 9</b>: Scatter plot for T7 based optimized iFFL system (forward scatter vs side scatter)]] </li>
 +
</center>
 +
    </ul></div>  
  
 
===References===
 
===References===
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<ul>
 
<ul>
 
<li>
 
<li>
<a id="Lou2012" href="https://www.nature.com/articles/nbt.2401" target="_blank">
+
<a id="Doyle2013" href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082120" target="_blank">
Lou, C., Stanton, B., Chen, Y.-J., Munsky, B., & Voigt, C. A. (2012). Ribozyme-based insulator parts buffer synthetic circuits from genetic context. <i>Nature Biotechnology</i>, 30(11), 1137–1142.</a>
+
Doyle, E. L., Hummel, A. W., Demorest, Z. L., Starker, C. G., Voytas, D. F., Bradley, P., & Bogdanove, A. J. (2013). TAL Effector Specificity for base 0 of the DNA Target Is Altered in a Complex, Effector- and Assay-Dependent Manner by Substitutions for the Tryptophan in Cryptic Repeat –1. <i>PLoS ONE</i>, 8(12).</a>
 
</li>
 
</li>
 +
 
</ul>
 
</ul>
 
</html>
 
</html>

Latest revision as of 20:56, 6 December 2019

T7 promoter based optimized iFFL

Genetic implementation of an incoherent feed-forward loop (iFFL) in which a weak strength T7 promoter with a binding site for TALE is controlling GFP expression.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 250
    Illegal XhoI site found at 3335
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
    Illegal AgeI site found at 1277
  • 1000
    COMPATIBLE WITH RFC[1000]

The two transcriptional units in this composite part are oriented outwards.

Usage and Biology

An incoherent feed-forward loop (iFFL) is a unique control systems motif where the output signal is robust to changes in the input signal (as shown in Figure 1). This is achieved by the introduction of a repressor.

  • Figure 1: Overview of incoherent feed-forward loop

iFFL can be applied to genetic circuits to achieve expression independent from copy number, transcriptional and translational rates. To implement the iFFL in a genetic circuit, TALE proteins can be used. These proteins consist of repeats where 12th and 13th amino acids can vary, these are called the repeat variable diresidue (RVD). RVDs have been shown to bind to DNA in a simple one-to-one binding code (Doyle, Stoddard et al., 2013). The direct correspondence between amino acids allows scientists to engineer these repeat regions to target any sequence they want. In our system, we used the TALE protein as a repressor by engineering promoters to contain the binding site of this specific TALE protein (T7sp1 promoter and PBHRsp1 promoter).
In our genetic circuit, a unrepressed promoter controls the expression of TALE protein while the promoters with the TALE binding sites drive expression of GFP.

  • Figure 2: Overview of how the TALE proteins represses GFP

When transcriptional units are placed in series due to low effieciency of terminators, leaky expression of the gene in the neighboring transcriptonal unit can occur. This significantly influences the behavior of the iFFL genetic circuit as predicted by our model. Hence, the transcriptional unit of TALE is oriented in a different orientation than the transcriptional unit of GFP (as shown in Figure 2).

An interesting application of the iFFL is to achieve controllable gene expression across different bacterial species. Gene expression in different bacterial contexts is influenced by changes in copy number, transcriptional and translational rates. To achieve expression robust to changes in transcriptional and tranlational rates, the ratio of transcriptional and translational rates of GFP and repressor need to be constant. This can be achieved by using orthogonal T7 promoter and its variants (T7 promoter, Weak T7 promoter, Medium T7 promoter, Medium T7sp1 promoter and T7sp1 promoter).

Apart from being able to achieve stable gene expression across different bacterial species, it is necessary to attain different levels of gene expression. The T7 promoter based optimized iFFL can be used to obtain higher levels of gene of interest (GFP) expression as the expression of TALE is driven by a lower strength promoter (Weak T7 promoter) compared to the promoter (Medium T7sp1 promoter) driving GFP expression.

Strain Construction

The construct was assembled by golden gate assembly based modular cloning system. First, the individual transcriptional units were cloned into level 1 destination vectors pICH47761 and pICH47822 by BsaI based golden gate assembly. The multi-transcriptional unit construct was assembled by a BpiI based golden gate. The assembly was a one-pot restriction-ligation reaction where the individual level 1 constructs were added along with the destination vector pICH48055. The correct clone was distinguished by blue-white screening and the construct was confirmed by sequencing. The cloning protocol can be found in the MoClo section below.

Modular Cloning

Modular Cloning (MoClo) is a system which allows for efficient one pot assembly of multiple DNA fragments. The MoClo system consists of Type IIS restriction enzymes that cleave DNA 4 to 8 base pairs away from the recognition sites. Cleavage outside of the recognition site allows for customization of the overhangs generated. The MoClo system is hierarchical. First, basic parts (promoters, UTRs, CDS and terminators) are assembled in level 0 plasmids in the kit. In a single reaction, the individual parts can be assembled into vectors containing transcriptional units (level 1). Furthermore, MoClo allows for directional assembly of multiple transcriptional units. Successful assembly of constructs using MoClo can be confirmed by visual readouts (blue/white or red/white screening). For the protocol, you can find it here.


Note: The basic parts sequences of the Sci-Phi 29 collection in the registry contain only the part sequence and therefore contain no overhangs or restriction sites. For synthesizing MoClo compatible parts, refer to table 2.


Table 1: Overview of different level in MoClo

Level Basic/Composite Type Enzyme
Level 0 Basic Promoters, 5’ UTR, CDS and terminators BpiI
Level 1 Composite Transcriptional units BsaI
Level 2/M/P Composite Multiple transcriptional units BpiI

For synthesizing basic parts, the part of interest should be flanked by a BpiI site and its specific type overhang. These parts can then be cloned into the respective level 0 MoClo parts. For level 1, where individual transcriptional units are cloned, the overhangs come from the backbone you choose. The restriction sites for level 1 are BsaI. However, any type IIS restriction enzyme could be used.


Table 2: Type specific overhangs and backbones for MoClo. Green indicates the restriction enzyme recognition site. Blue indicates the specific overhangs for the basic parts

Basic Part Sequence 5' End Sequence 3' End Level 0 backbone
Promoter NNNN GAAGAC NN GGAG TACT NN GTCTTC NNNN pICH41233
5’ UTR NNNN GAAGAC NN TACT AATG NN GTCTTC NNNN pICH41246
CDS NNNN GAAGAC NN AATG GCTT NN GTCTTC NNNN pICH41308
Terminator NNNN GAAGAC NN GCTT CGCT NN GTCTTC NNNN pICH41276

Characterization

According to our model, the genetic implementation of the iFFL system yields the same gene of interest (GOI) expression levels when the transcriptional rate of both genes (TALE and GOI) are changed in the same ratio as can be seen in Figure 3.

  • Figure 3: Steady-state GFP production while transcriptional rates of both TALE and GOI are changed. The lines indicate constant ratio of transcriptional rates

For the system to be versatile, it is important to achieve different levels of output gene expression (GFP). Hence, in the optimized iFFL system the TALE repressor is expressed by a higher strength promoter compared to the promoter controlling GFP expression.

To validate the insulation of the gene expression (GFP) in the iFFL system to changes in transcriptional rates, GFP fluorescence was measured at different IPTG concentrations by flow cytometry at log-phase of growth. Change in IPTG concentrations, changes in vivo concentrations of T7 RNAP and this contributes to variations in transcriptional rates. In unrepressed systems, the expression of the GOI is a function of IPTG concentrations. However, in iFFL systems due to the expression of TALE repressor, similar GFP expression across different IPTG concentrations is expected.

The iFFL system was validated by measuring GFP using flow cytometry. As a control, GFP under the control of T7sp1 promoter was used. As a reference for background fluorescence, E. coli BL21(DE3) cells without plasmids were used. The protocol for sample preparation can be found here.

Scatter plots (Figures 7 and 8) for the unprepressed control and T7 based optimized iFFL were gated by eye to select for the most dense regions. Figures 4 and 5 show fluorescence measurements for the gated populations of the unrepressed control and optimized iFFL system at different IPTG concentrations respectively.

  • Figure 4: Fluorescence measurements from gated population of unrepressed control (for scatter plot refer Figure 7) at different IPTG concentrations. Black: E. coli BL21(DE3)(Green: 0mM, Red: 0.1mM, Blue: 0.5mM and Purple: 1mM
  • Figure 5: Fluorescence measurements from gated population of optimized iFFL system (for scatter plot refer Figure 8) at different IPTG concentrations. Black: E. coli BL21(DE3), Green: 0mM, Red:0.1mM, Blue: 0.5mM and Purple: 1mM

In Figure 4, it is seen that GFP fluorescence of the unrepressed control changes with IPTG concentrations. However,(in Figure 5) for the optimized iFFL system, the GFP fluorescence curves for all the IPTG concentrations overlap and therefore is independent of IPTG concentrations.

  • Figure 6: Steady state GFP fluorescnece measurements using flow cytometry at different IPTG concentrations. Measurements were made at log-phase of growth and the median fluorescence of the gated populations is plotted.

From Figure 6, it is seen that despite changes in IPTG the GFP fluorescence remains the same while the GFP fluorescence of the control increases with IPTG concentrations. This suggests that the gene of interest expression is independent of in-vivo T7 RNAP concentrations (IPTG concentrations) thereby independent of changes in transcriptional rate.

Conclusion

The characterization of the T7 based optimized iFFL system suggests that the circuit is robust to changes in transcriptional rates.


Raw Data

  • Figure 8: Scatter plot for unrepressed control (forward scatter vs side scatter)
  • Figure 9: Scatter plot for T7 based optimized iFFL system (forward scatter vs side scatter)

References