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| − | The composite part consists of cenA-hlyA-hlyb-hlyD, which expresses C. fimi endoglucanase CenA. | + | The composite part consists of cenA-hlyA-hlyB-hlyD, which expresses C. fimi endoglucanase CenA fused with HlyA, through α-hemolysin secretion system from uropathogenic E. coli, it can be secreted out of E. coli detected in the medium. |
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| − | The lacI gene is expressed from an unregulated promoter Placlq, and its product LacI represses the Plac promoter. Likewise, the Plac promoter controls the expression of the cI gene, and its product CI represses the PR promoter. The PR promoter controls the production of the red fluorescent protein (mRFP1), which represents the‘output’for the circuit, whereas the chemical inducer isopropyl β-D-thiogalactopyranoside (IPTG), which binds to LacI tetramers and renders them unable to repress Plac, provides an external control over the network and thus represents the‘input.’
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| | ===Usage and Biology=== | | ===Usage and Biology=== |
| − | The dual inverter consists of lactose operon, CⅠprotein and λPR012. All of the basic parts can be found in iGEM standard biological parts, but ECUST_iGEMers combined these basic parts to constitute a brand new composite part. | + | The secretory CenA consists of CenA (endoglucanase)and α-hemolysin secretion system. All of the basic parts can be found in iGEM standard biological parts, but ECUST_iGEMers combined these basic parts to constitute a new composite part. |
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| − | The LacI repressor is constitutively expressed which inhibits transcription from the pLac promoter in the absence of IPTG, thus λPR012 could be active. While the expression of the CI repressor can be induced by externally added IPTG and inhibits the transcription of λPR012, hence realizing the reversal of the function.
| + | By this part we can achieve secreting CenA out of E. coli, but the secretory efficiency was relatively low, and CenA expressed in E. coli has shown limited enzyme activity by our CMC-Na assay and literature results. Both reasons combined contribute to our expected but less satisfying results. |
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| | ===Characterization=== | | ===Characterization=== |
| − | In order to characterize PlacIq-LacI-Plac-cI-λPR dual inverter, ECUST_iGEMer constructed a dual-plasmid: pIN1-pIN2. | + | In order to characterize cenA-hlyA-hlyB-hlyD, ECUST_iGEMer inserted these genes after a modified pET28b--pIN2, a λ-driven expression and Kan-antibiotic vector . |
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| − | <div class="exper-com-box"><img style="width:500px;" src="https://static.igem.org/mediawiki/parts/4/49/T--ECUST_China--Figure.0_Gene_circuit_of_inverter_system.png"> <br><span style="font-size: 14px;"> | + | <div class="exper-com-box"><img style="width:500px;" src="https://2019.igem.org/File:T--ECUST_China--CenA.png"> <br><span style="font-size: 14px;"> |
| − | <b>Figure 0.</b> Gene circuit of inverter system</span></div></html> | + | <b>Figure 1.</b> Gene circuit of cenA-hlyA/hlyBhlyD/pIN2</span><br><span style="font-size: 12px;"><i>cex</i>: Cellulase exoglucanase gene, <i>cenA</i>: Cellulase endoglucanase gene,<i>hlyA, hlyB, hlyD</i>: α-hemolysin system gene</span></div></html> |
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| − | Before co-expressing this dual-plasmid, we verified the function of pIN1 and pIN2 respectively. pIN1 was composed of PlacIq-LacI-Plac-cI, using GFP as the reporter gene. Since CI and GFP were both under the control of Plac, the expression of GFP was disturbed by CI according to the measurement of GFP fluorescence. However, we performed SDS-PAGE to confirm that the pIN1 exactly worked.
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| − | <html><div class="exper-com-box"><img style="height:400px;" src="https://static.igem.org/mediawiki/parts/2/2a/T--ECUST_China--Figure.1_SDS-PAGE_results_of_pIN1.jpg"> <br><span style="font-size: 14px;">
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| − | <b>Figure 1.</b> SDS-PAGE results of pIN1</span></div> </html>
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| − | The SDS-PAGE results showed that for pIN1, cI (26.2 kDa) and Lac I (38.9 kDa) was successfully expressed after induction, indicating that the construction of pIN1 was preliminary successful.
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| − | pIN2 was relatively simple, just consisting of λPR012 and using mRFP as the reporter. In the absence of CI protein, λPR012 was constitutively active to express mRFP. So we could clearly recognized the positive colony via observing the color while construction (the red colony might be the positive one). After colony PCR and sanger sequencing, we incubated the positive colony in 5mL M9 containing 0.1% Kan at 37℃ for 12 hr.
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| − | <html><div class="exper-com-box"><img style="width:300px;" src="https://static.igem.org/mediawiki/parts/4/4c/T--ECUST_China--Figure.2_Plates_of_DH5α_transformants_%28pIN2%29.png"><br><span style="font-size: 14px;"> <b>Figure 2.</b> Plates of DH5α transformants (pIN2)</b></span></div>
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| − | <div class="exper-com-box"><img style="width:300px;" src="https://static.igem.org/mediawiki/parts/b/bb/T--ECUST_China--Figure.3_pIN2_growing_in_5mL_LB_for12_hr.png"><br><span style="font-size: 14px;"> <b>Figure 3.</b> pIN2 growing in 5mL LB for12 hr</b></span></div> </html>
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| − | Figure.3 showed clearly that after 12 hours’ growing in M9, mRFP of pIN2 was highly expressed. In order to characterize the function of pIN2, we set the negative control (pET) and experiment group (pIN2), both growing in 5mL M9 (containing 0.1% Kan) at 37℃ for 12 hr and then being transferred to the secondary culture. We took samples from the secondary culture in 0h, 1h, 2h and 3h and measured the samples’ fluorescence.
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| − | <html><div class="exper-com-box"><img style="width:400px;" src="https://static.igem.org/mediawiki/parts/d/db/T--ECUST_China--Figure.4_Fluorescence_intensity_of_mRFP.jpg"> <br><span style="font-size: 14px;">
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| − | <b>Figure 4. </b>Fluorescence intensity of mRFP</span></div> </html>
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| − | The red fluorescence in pIN2 was extremely high and stable, while the red fluorescence of negative control (pET) was barely detectable, conforming that pIN2 worked effectively.
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| − | After charactering the pIN1 and pIN2 seperately.The whole circuit PlacIq-LacI-Plac-cI-λpR (pIN1-pIN2) measurement was performed in liquid cultures. pIN1 and pIN2 were initially co-transferred to E.coli DH5α competent cell. The positive colony was then grown in 5mL M9 at 37℃ for 12 hr, then transferred to 100 mL M9 for enlarge cultivation. After the cells growing to OD600=0.5, we added different concentration of IPTG(0.1μM,1μM, 10μM, 100μM, 1000μM ).
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| − | <html><div class="exper-com-box"><img style="width:400px;" src="https://static.igem.org/mediawiki/parts/b/be/T--ECUST_China--Figure.5_Liquid_M9_media_in_15hr_of_dual-plasmid_%28pIN1_%2B_pIN2%29_in_different_concentraton_of_IPTG..jpg"> <br><span style="font-size: 14px;"> <b>Figure 5.</b> Liquid M9 media in 15hr of dual-plasmid (pIN1 + pIN2) in different concentraton of IPTG</span></div> </html>
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| − | We took samples of each IPTG concentration before and after induction (almost 16hr after induction). The samples were then transferred to a 96-well microplate in which mRFP fluorescence (590 nm excitation, 645 nm emission, top 50% cutoff) was measured by using a fluorescence microplate reader. The fluorescence data were normalized against cell densities which were measured by using a microplate reader at 600 nm.
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| − | <html><div class="exper-com-box"><img style="width:400px;" src="https://static.igem.org/mediawiki/parts/8/80/T--ECUST_China--Figure.6_Fluorescence_intensity_induced_by_different_concentrations_of_IPTG.jpg"> <br><span style="font-size: 14px;">
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| − | <b>Figure 6.</b> Fluorescence intensity induced by different concentrations of IPTG</span> </div></html>
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| − | Measurements of the circuits in response to varying IPTG levels were summarized in the transfer curve, where each point on the curve represented fluorescence data from three independent cultures of the same circuit under the same induction conditions.
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| − | Just as shown in the figure, as the concentration of IPTG rose, the expression of mRFP decreased, indicating that the dual-plasmid worked effectively. The LacI protein was constitutively expressed from PlacIQ and repressed the Plac promoter. Plac transcriptional activity was controlled by modulating the concentration of an externally added inducer, IPTG. The expression of the CI repressor was controlled by Plac . Repressor CI acts on PλRO12 on pIN2 to repress the transcription of the mRFP gene, the output fluorescence indicator. So without IPTG, the CI level was low and mRFP level was high; adding IPTG increased CI levels and in turn decreased mRFP levels. To sum up, ECUST_China iGEMers quantitatively characterized PlacIq-LacI-Plac-cI-λpR dual inverter successfully
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| | ===References=== | | ===References=== |
The composite part consists of cenA-hlyA-hlyB-hlyD, which expresses C. fimi endoglucanase CenA fused with HlyA, through α-hemolysin secretion system from uropathogenic E. coli, it can be secreted out of E. coli detected in the medium.
The secretory CenA consists of CenA (endoglucanase)and α-hemolysin secretion system. All of the basic parts can be found in iGEM standard biological parts, but ECUST_iGEMers combined these basic parts to constitute a new composite part.
By this part we can achieve secreting CenA out of E. coli, but the secretory efficiency was relatively low, and CenA expressed in E. coli has shown limited enzyme activity by our CMC-Na assay and literature results. Both reasons combined contribute to our expected but less satisfying results.
In order to characterize cenA-hlyA-hlyB-hlyD, ECUST_iGEMer inserted these genes after a modified pET28b--pIN2, a λ-driven expression and Kan-antibiotic vector .
[1]Yokobayashi Y, Weiss R, Arnold FH. Directed evolution of a genetic circuit. Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16587-91. Epub 2002 Nov 25. PubMed PMID: 12451174; PubMed Central PMCID: PMC139187.
