Difference between revisions of "Part:BBa K3093008"
(→‎References)
 
(13 intermediate revisions by 2 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K3093008 short</partinfo>
 
<partinfo>BBa_K3093008 short</partinfo>
  
−
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.
−
 
+
−
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.’
+
  
 
===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.
  
−
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.
  
 
===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.
 +
α-hemolysin specific signal peptide HlyAs, which corresponds to residues 965-1024 of α-hemolysin, was fused to the C-terminus of cenA and cex by overlapping PCR. In addition, the genes encoding the components of translocator, hlyB and hlyD, were amplified together by PCR using part:BBa_K1166002 as template, cenA was amplified by PCR using part:BBa_K523015. Using seamless cloning we linkers thes three fragments together,it was then transformed into E. coli strain DH5α and positive clones were selected by Kan antibiotics.  
  
−
<html><style>
+
<html><img style="width:300px;padding-left:200px;" src="https://2019.igem.org/wiki/images/a/a6/T--ECUST_China--CenA.png"> <br><span style="font-size: 14px;">  
−
.exper-com-box{
+
  <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>
−
width: 80%;
+
−
text-align: center;
+
−
padding-bottom: 20px;
+
−
}</style>
+
−
<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;">  
+
−
  <b>Figure 0.</b> Gene circuit of inverter system</span></div></html>  
+
  
−
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.
+
Positive clones were further inoculated overnight in 4 mL LB broth with proper antibiotic concentration. The next day, we transferred 1 mL culture to 100mL LB in a 37℃ shaker proper antibiotics added. For approximately 3hrs, O.D. of cell growth was measured, until it reached 0.6, we placed the shaker in 30℃ at 220rpm for induce. After 8 hours, the whole culture medium was spun down at 4000rpm for 10 minutes at 4℃. After that, we further used centrifugation of 1200rpm for 10 minutes at 4℃ to  further discard all non-soluble parts of cells and supernatant was collected. Next, we applied a 3kDa protein concentration tube to concentrate the supernatant by 20 times for better visualization. The concentrated solution was used as crude CenA solution.  
−
   
+
−
<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;">
+
−
<b>Figure 1.</b> SDS-PAGE results of pIN1</span></div> </html>
+
−
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.
+
  
−
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.  
+
The CMCNa assays were incubated at 37°C and pH7. Based on literature, In a microtube 18μL of 1% CMC-Na was added, followed by 2μL crude CenA and the mix was incubated for 1 hour. To stop and reveal the reaction, 30 μL of DNS were added, after 1 hour of 99°C incubation, absorbance at 540 nm was determined. For negative control, we performed contrast reactions at the same time, but it was reacted at the temperature below 4°C so that the enzyme activity was inhibited.  
  
−
<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>
+
<html><img style="width:500px;padding-left:100px;" src="https://2019.igem.org/wiki/images/b/be/T--ECUST_China--super1.png"> <br><span style="font-size: 14px;">  
−
<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>
+
  <b>Figure 2.</b> supernatant enzyme activity of cenA-hlyA/hlyBhlyD/pIN2</span><br><span style="font-size: 12px;">The supernatant was concentrated by 20 times when performing CMCNa assay, but the enzyme activity had been adjusted to standard unit</span></div></html>
−
+
−
 
+
−
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.
+
−
 
+
−
 
+
−
<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;">  
+
−
  <b>Figure 4. </b>Fluorescence intensity of mRFP</span></div> </html>
+
−
 
+
−
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.
+
−
 
+
−
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 ).
+
−
 
+
−
<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>
+
−
 
+
−
 
+
−
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.
+
−
 
+
−
<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;">
+
−
<b>Figure 6.</b> Fluorescence intensity induced by different concentrations of IPTG</span> </div></html>
+
−
 
+
−
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.
+
−
 
+
−
 
+
−
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
+
  
 
===References===
 
===References===
−
[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.
+
[1]Duedu KO, French CE. Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass. Enzyme Microb Technol. 2016 Nov;93-94:113-121. doi: 10.1016/j.enzmictec.2016.08.005. Epub 2016 Aug 8. PubMed PMID: 27702471.
  
 +
[2]Su L, Chen S, Yi L, Woodard RW, Chen J, Wu J. Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3). Microb Cell Fact. 2012 Jan 12;11:8. doi: 10.1186/1475-2859-11-8. PubMed PMID: 22239833; PubMed Central PMCID: PMC3286373.
  
−
<!-- Add more about the biology of this part here
 
−
===Usage and Biology===
 
  
−
<!-- -->
 
−
<span class='h3bb'>Sequence and Features</span>
 
−
<partinfo>BBa_K3093100 SequenceAndFeatures</partinfo>
 
−
 
−
 
−
<!-- Uncomment this to enable Functional Parameter display
 
−
===Functional Parameters===
 
−
<partinfo>BBa_K3093100 parameters</partinfo>
 
−
<!-- -->
 
−
 
−
 
−
 
−
 
−
<!-- Add more about the biology of this part here
 
−
===Usage and Biology===
 
  
 
<!-- -->
 
<!-- -->

Latest revision as of 11:17, 21 October 2019


cenA-hlyA+secretion system

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.

Usage and Biology

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.

Characterization

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. α-hemolysin specific signal peptide HlyAs, which corresponds to residues 965-1024 of α-hemolysin, was fused to the C-terminus of cenA and cex by overlapping PCR. In addition, the genes encoding the components of translocator, hlyB and hlyD, were amplified together by PCR using part:BBa_K1166002 as template, cenA was amplified by PCR using part:BBa_K523015. Using seamless cloning we linkers thes three fragments together,it was then transformed into E. coli strain DH5α and positive clones were selected by Kan antibiotics.


Figure 1. Gene circuit of cenA-hlyA/hlyBhlyD/pIN2
cex: Cellulase exoglucanase gene, cenA: Cellulase endoglucanase gene,hlyA, hlyB, hlyD: α-hemolysin system gene

Positive clones were further inoculated overnight in 4 mL LB broth with proper antibiotic concentration. The next day, we transferred 1 mL culture to 100mL LB in a 37℃ shaker proper antibiotics added. For approximately 3hrs, O.D. of cell growth was measured, until it reached 0.6, we placed the shaker in 30℃ at 220rpm for induce. After 8 hours, the whole culture medium was spun down at 4000rpm for 10 minutes at 4℃. After that, we further used centrifugation of 1200rpm for 10 minutes at 4℃ to further discard all non-soluble parts of cells and supernatant was collected. Next, we applied a 3kDa protein concentration tube to concentrate the supernatant by 20 times for better visualization. The concentrated solution was used as crude CenA solution.

The CMCNa assays were incubated at 37°C and pH7. Based on literature, In a microtube 18μL of 1% CMC-Na was added, followed by 2μL crude CenA and the mix was incubated for 1 hour. To stop and reveal the reaction, 30 μL of DNS were added, after 1 hour of 99°C incubation, absorbance at 540 nm was determined. For negative control, we performed contrast reactions at the same time, but it was reacted at the temperature below 4°C so that the enzyme activity was inhibited.


Figure 2. supernatant enzyme activity of cenA-hlyA/hlyBhlyD/pIN2
The supernatant was concentrated by 20 times when performing CMCNa assay, but the enzyme activity had been adjusted to standard unit

References

[1]Duedu KO, French CE. Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass. Enzyme Microb Technol. 2016 Nov;93-94:113-121. doi: 10.1016/j.enzmictec.2016.08.005. Epub 2016 Aug 8. PubMed PMID: 27702471.

[2]Su L, Chen S, Yi L, Woodard RW, Chen J, Wu J. Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3). Microb Cell Fact. 2012 Jan 12;11:8. doi: 10.1186/1475-2859-11-8. PubMed PMID: 22239833; PubMed Central PMCID: PMC3286373.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 114
    Illegal NotI site found at 1258
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1171
    Illegal BamHI site found at 62
    Illegal BamHI site found at 307
    Illegal XhoI site found at 71
    Illegal XhoI site found at 669
    Illegal XhoI site found at 918
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 449
    Illegal NgoMIV site found at 1374
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 353