Team:Tianjin/Project/Design

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                <a href="https://2013.igem.org/Team:Tianjin/Project">Overview</a>
 
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                <a href="https://2013.igem.org/Team:Tianjin/Project/Background">Background</a>
 
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                <a href="https://2013.igem.org/Team:Tianjin/Project/Design">Design</a>
 
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                <a href="https://2013.igem.org/Team:Tianjin/Project/Experiment">Experiment&Result</a>
 
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                        <a href="https://2013.igem.org/Team:Tianjin/Project/Experiment">System construction and optimization</a>
 
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                        <a href="https://2013.igem.org/Team:Tianjin/Project/Experiment/Endo-Induce">Endogenous Alkanes Induce</a>
 
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                        <a href="https://2013.igem.org/Team:Tianjin/Project/Experiment/Exo-Induce">Exogenous Alkanes Induce</a>
 
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                        <a href="https://2013.igem.org/Team:Tianjin/Project/Experiment/TetA">Endogenous Induce&Resistance Selection</a>
 
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                    <li>
 
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                        <a href="https://2013.igem.org/Team:Tianjin/Project/Experiment/LibraryConstruction">Library Construction</a>
 
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<center><span style="font-family:Arial;font-size:46px;color:#000;"> Design </span></center>
 
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= ALKR-PalkM system in the original organism=
 
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<div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/3/31/ALKR-PalkM_in_original_origanism.png" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/3/31/ALKR-PalkM_in_original_origanism.png" width="600px" /></a></div>
 
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<b>Figure 1.</b>&nbsp; Mechanism of ALKR-PalkM system in <i>Acinetobacter baylyi </i>ADP1</div>
 
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<div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/4/40/Tju-bg-03.gif" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/4/40/Tju-bg-03.gif" width="420px" /></a></div>
 
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<b>Figure 2.</b>&nbsp; Homologous modeling figure of protein ALKR </div>
 
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<p> Protein ALKR and promoter alkM are originally found from <i>Acinetobacter baylyi</i> ADP1. The ALKR-PalkM gene circus is part of the regulation system of alkane metabolism.</p>
 
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<p><i> Acinetobacter </i>sp. are ubiquitous bacteria in natural aquatic and soil environment that are frequently found to be capable to degrade a broad range of carbon chain alkenes and alkanes[3]. And <i>Acinetobacter baylyi</i> ADP1 is able to use long-chain-length alkanes with at least 12 carbon atoms as the sole source of carbon and energy[4]. In <i>Acinetobacter baylyi</i> ADP1, P-alkM is the promoter of alkane hydroxylase genes which encodes alkane hydroxylase(alkM), a crucial enzyme in the degradation of alkanes. AlkM, together with rubredoxin (RubA) and rubredoxin reductase(RubB) forms a three-component alkane monooxygenase complex which oxidate inert alkane to the respective primary alcohol[4]. AlkR is an AraC/XylS-like transcriptional regulatory protein. It includes C-terminal DNA-binding domain for promoter binding and N-terminal domain for inducer recognition[2]. Its function is to recognize alkane molecule after its own dimerization and induce promoter alkM. The activity of alkane hydroxylase could impede ALKR’s function. When alkane molecules occur, they could be recognized by protein ALKR. A three component complex is formed. The inducer complex can bind with promoter alkM and activate the genes in the downstream of PalkM.</p>
 
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= Our first trail=
 
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<p> Our goal is to build a specific alkane sensing device in the cell. The device could sense alkanes and transfer the inconspicuous alkanes to some conspicuous signals, such as some chromophoric or florescent signals. We could place both alkane biosynthesis module and AlkSensor into E coli. The alkane molecules produced by alkane biosynthesis module can be recognised by AlkSensor. And AlkSensor generate a conspicuous signal which can be easily detected.</p>
 
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<div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/d/d2/Tju-A_rough_idea_of_our_design.png" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/d/d2/Tju-A_rough_idea_of_our_design.png" width="600px" /></a></div>
 
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<b>Figure 3.</b>&nbsp; A rough idea of our design </div>
 
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<p> Based on the ALKR-PalkM mechanism in <i>Acinetobacter baylyi</i> ADP1, we designed an alkane sensor in E coli. The sensor is composed of two parts: alkane sensing part and signal generation part. The core of alkane sensing part in <i>Acinetobacter baylyi</i> ADP1 is promoter alkM, so we took PalkM into our design. Besides, PalkM must co-function with transcription factor ALKR, so we constructed ALKR into the sensor. ALKR and PalkM are sufficient for sensing alkanes. The function of signal generation part is to output a conspicuous or easily detectable signal. In this case, we chose RFP as the reporter.</p>
 
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<p> Our first design is shown in figure 3. We linked ALKR with a constitutive promoter,a strong promoter J23100, and linked RFP with P-alkM. AlkR is followed by a short terminator B1006.The two gene pieces are constructed into plasmid pSB1C3. The inputs of AlkSensor are alkanes and the outputs are RFPs.</p>
 
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<b>Figure 4.</b>&nbsp; The first version of AlkSensor </div>
 
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= Optimization and our final construction=
 
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<p> We performed a blank test on the first version of AlkSensor. Without any inducers, AlkSensor still shown significant leakage. We can see from the results that many of the bacterial colonies are red enough to be distinguished by naked eyes.</p>
 
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<b>Figure 5.</b>&nbsp; The leakage of the first version of AlkSensor is non-ignorable </div>
 
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<p> Followings are three possible causes of the leakage.</p>
 
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<p> a) Promoter alkM has some leakage, to be more specific, it binds to RNA polymerase without undergoing a conformation change and activates the transcription</p>
 
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<p> b)Protein ALKR or dimerized ALKR could induce promoter alkM without alkane molecules.</p>
 
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<p> c)The terminator upstream of PalkM is BBa_B1006 which is relatively short (only 34 bp), and it has a weak terminating ability, while the constitutive promoter BBa_J23100 upstream of AlkR is strong, so expression of RFP might be influenced.</p>
 
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<p> We Reconstruct AlkSensor :</p>
 
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<p> a) Changed the promoter of ALKR to a weaker promoter.</p>
 
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<p> b) Switch the position of  gene pieces ALKR and PalkM-RFP </p>
 
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<p> c) Replace terminator B1006 with a stronger terminator, B0015 </p>
 
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<b>Figure 6.</b>&nbsp; AlkSensor after reconstruction </div>
 
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<p> Through another blank test we found that the reconstruction could decrease AlkSensor’s leakage significantly. After optimization, the percent of bacterial colonies are red enough to be distinguished by naked eyes decrease from 95% to 15%.</p>
 
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<b>Figure 7.</b>&nbsp; Comparison of two versions of AlkSensor </div>
 
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<p> We also used full dose octane to induce the two sensors. After a certain period of time of cultivation, the relative fluorescent intensity was measured. The results show that after reconstruction the leakage decreases by 50%, the dynamic range increases by 5 folds, which means that the optimized AlkSensor has stronger ability to distinguish different inputs.</p>
 
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<b>Figure 8.</b>&nbsp; Octane induce test of two versions of AlkSensor </div>
 
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Latest revision as of 02:27, 29 October 2013

Retrieved from "http://2013.igem.org/Team:Tianjin/Project/Design"