Difference between revisions of "Part:BBa K3089034"
 
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<partinfo>BBa_K3089034 short</partinfo>
 
<partinfo>BBa_K3089034 short</partinfo>
  
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T7 promoter+csgA-linker-mfp5-linker-sfGFP, sfGFP as marker
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<!-- Add more about the biology of this part here
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This composite part is meant to express csgA-linker-mfp5-linker-sfGFP fusion genes under T7 promoter. CsgA is an amyloid-like protein encoded on genome of E.coli MG1655 providing mechanical cohesive strength. Mfp5 is mussel foot proteins from Mytilus galloprovincialis responsible for interface adhesion. Compared to T7 promoter+csga-linker-mfp5-linker-His( <a href="https://parts.igem.org/Part:BBa_K3089021"target="_blank">BBa_K3089021</a>), we have added sfGFP to characterize the expression of the recombinant protein. It is a robustly folded version of GFP, called ‘superfolder’ GFP, that folds well even when fused to poorly folded polypeptides (Waldo et al, 2006).
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'><h3>Sequence and Features</h3></span>
 
<partinfo>BBa_K3089034 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3089034 SequenceAndFeatures</partinfo>
  
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<h3>Characterization</h3>
  
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<h3>Introduction</h3>
 
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<p>
 
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This composite part is meant to express csgA-linker-mfp5-linker-sfGFP fusion genes under the T7 promoter. CsgA is an amyloid-like protein encoded on genome of E.coli MG1655 providing cohesive mechanical strength.
 
  
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Mfp5 is mussel foot proteins from Mytilus galloprovincialis responsible for interface adhesion. Compared to  T7 promoter+csga-linker-mfp5-linker-His(BBa_K3089021), we have added sfGFP to characterise the expression of the recombinant protein. It is a robustly folded version of GFP, called ‘super folder’ GFP, that folds well even when fused to poorly folded polypeptides (Waldo et al., 2006).
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</p>
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<center><Figure>
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<img width="600px" src="https://2019.igem.org/wiki/images/5/52/T--Greatbay_SCIE--P--034-Figure_1.png">
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</figure></center>
  
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<h3>Characterization</h3>
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<center><figcaption> The circuit of the protein BBa_K30889034 </figcaption></center>
  
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<h3>Fluorescence analysis</h3>
  
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<h3> Protein expression </h3>
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<p>
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T7 promoter+csgA-linker-mfp5-linker-sfGFP was cloned into pET28b and transformed into E.coli BL21 (DE3).We grew 25-ml cultures of
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<i>E. coli BL21</i>
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(DE3) bearing csgA-linker-mfp5-linker-sfGFP in LB medium containing kanamycin (50 mg/ml) overnight. We grew 1000-fold dilutions in 200-μL cultures to ~0.2/0.5/0.8 OD600 nm in a 96-well plate with cover and induced them at 37℃ with 500μM IPTG for 22 h.
  
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<html>
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OD600nm and fluorescence were measured (488-nm excitation, 530-nm emission,10-nm bandpass for GFP) with a Microplate Fluorescence Reader (THERMO Varioskan Flash). Fluorescence was normalised by dividing by the OD600 nm. We continuously monitored the OD600nm and fluorescence of these four strains and plotted the graph for their growth and induced fluorescence. We added IPTG to these strains at different times of their log phase, such as OD600nm=0.2（early）, 0.5（medium）, 0.8（late）.   
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<img width="60
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0px" src="https://2019.igem.org/wiki/images/5/52/T--Greatbay_SCIE--P--034-Figure_1.png">
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</figure>
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<figcaption> The circuit of the protein BBa_K30889034 </figcaption>
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The predicted size of rBalcp19k-linker-mfp5 is 28.17 kDa, and the isoelectric point is 10.41. rBalcp19k-linker-mfp5 was cloned into pET28b and expressed in E.coli BL21(DE3) Rosetta by 500μM IPTG for 5h at 37℃.
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Results were measured by the ratio of fluorescence to OD600nm. CsgA-Mfp5-sfGFP had a relatively much poorer expression compare with sfGFP, which was used as control (Figure 1ABC).  
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To detect its expression, whole cells were collected after induction by centrifuging and prepared for SDS-PAGE. Results showed that no protein bands of rBalcp19k-linker-mfp5(~28 kDa) could be observed on lane rBalcp19k-mfp5 compared with lane pET28b (pET28b empty vector)(Figure 1A), which means the expression of this protein is not well in BL21(DE3) Rosetta.
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We can get the same results using quantitative densitometry analysis of SDS-PAGE gels (Figure 1B).
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</p>  
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<center><figure>
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<img width="450px" src="https://2019.igem.org/wiki/images/5/52/T--Greatbay_SCIE--P--034-Figure_1.png">
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<img width="600px" src="https://2019.igem.org/wiki/images/8/8f/T--Greatbay_SCIE--Fluorescence_analysis.png">
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</figure>
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</figure></center>
  
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<figcaption> Figure 2 Detection of the expression level of all recombinant proteins by SDS-PAGE.(A) SDS-PAGE of whole-cell lysates of all recombinant proteins. Red arrows show the predicted place of certain proteins. (B) Protein SDS-PAGE bands optical densities were measured by quantitative densitometry of SDS-PAGE of whole-cell aliquots. </figcaption>
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<center><figcaption> Figure 2. Normalised fluorescence by dividing with OD600nm of sfGFP fused constructs（A-C）、OD600m(D-F) and fluorescence(G-I). Arrows indicate timing to add IPTG. Six repeats were monitored for each group and anomalies below 0 was ignored. </figcaption></center>
  
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<h3> Protein purification </h3>
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<center><figure>
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<img width="450px" src="https://2019.igem.org/wiki/images/4/4e/T--Greatbay_SCIE--Normalized_fluorescence_measurement_of_CsgA-linker-Mfp5-linker-sfGFP.png">
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</figure></center>
  
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<p>
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<center><figcaption> Figure 3. Normalized fluorescence (Fluorescence/OD600nm) measurement of CsgA-linker-Mfp5-linker-sfGFP. 500uM IPTG was added into cultures when it reached OD600nm at 0.2, 0.5, 0.8 separately after a 1000-fold dilution from overnight cultures. Arrows indicate timing to add IPTG. A.U. (arbitrary units) calculated by dividing fluorescence with OD600nm value. Six repeats were monitored for each group and anomalies below 0 was ignored. </figcaption></center>
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Barnacle cement proteins are very promising in making biomedical bio-glues. rBalcp19K from Balanus albicostatus had the properties of both self-assembly and adhesion.
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It also could function in more basic condition than Mfps. Thus we also designed a novel recombinant protein by combining it with Mfp5. We expected rBalcp19k-Mfp5 would perform better adhesive ability to solidify our idea of modularisation of Mfp5.
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We tried to purify it under native conditions, and we found bands of rBalcp19K-linker-mfp5 appeared between 25kDa and 35kDa on 12% SDS-PAGE gel(Figure 2), which meant it was successfully expressed and purified under native condition(see details on our methods).  
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Protein concentrations of rBalcp19k-linker-mfp5 were measured by BCA assay, and its yield is 1mg/L.
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<center><figure>
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<Figure>
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<img width="450px" src="https://2019.igem.org/wiki/images/f/f8/T--Greatbay_SCIE--Fluorescence_curve_of_CsgA-linker-Mfp5-linker-sfGFP.png">
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<img width="450px" src="https://static.igem.org/mediawiki/parts/d/d9/T--Greatbay_SCIE--SDS-PAGE_of_rBalcp19k-mfp5.png">
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</figure></center>
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</figure>
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<figcaption>  
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<center><figcaption> Figure 4. Fluorescence curve of CsgA-linker-Mfp5-linker-sfGFP </figcaption></center>
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Figure 3. SDS-PAGE of purified rBalcp19k-mfp5 by affinity chromatography under native conditions.
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Lanes: M, protein molecular weight marker; NC, whole-cell sample of pET28b empty vector; WC, whole-cell sample of recombinant protein rBalcp19K; S, soluble cell fraction; W1, fraction.
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</figcaption>
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<h3> Surface coating analysis </h3>
 
 
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After obtaining a small number of recombinant proteins, surface coating analysis for qualitatively assessing the surface adsorption ability of recombinant proteins was conducted on two of most commonly used bio-related surfaces: hydrophilic glass slides and hydrophobic polystyrene tissue culture plates.
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Interestingly,  
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As shown in Figure3, rBalcp19k-linker-mfp5 recombinant protein showed higher surface absorption abilities on both different substrates than rBalcp19k without fusion of mfp5 on its C-terminal.  
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<b>adding inducer in early stage of log phase (OD=0.2) would delay the growth of CsgA-linker-Mfp5-linker-sfGFP (Figure 1D), letting them enter to lag phase again.</b>
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It’s suggested that Mfp improves the coating ability of rBalcp19k-linker-mfp5 fusion proteins. The In-vitro DOPA modification by mTyr-CNK tyrosinase significantly improved its surface absorption abilities, which suggested the positive contribution of DOPA in adhesive protein performances.
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During this second-lag-phase, the fluorescence was growing continuously (Figure 2G), which means proteins was still accumulating in cells. However, when growth entered log phase again, the normalised fluorescence reduced to some extent due to the rapid increase of OD600nm value.
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<b> These phenomena are generally not observed when adding IPTG in medium (OD=0.5) and late log phase(OD=0.8)( Figure 1EF). </b>
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For what we concern is the total yield, in other words, the absolute fluorescence of the culture. Results showed adding IPTG in early log phase significantly reduced overall expression, and the fluorescence reached its peak in 5 hours (Figure 4).
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</p>
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<p>
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<b>We concluded that we should add inducer in late log phase for higher-level expression and 5 hours’ induction was enough to get a highest yield.</b>
 
</p>
 
</p>
  
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<Figure>
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<h3>Reference</h3>
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<img width="450px" src="https://static.igem.org/mediawiki/parts/8/8e/T--Greatbay_SCIE--P--Surface_coating.jpeg">
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<p>
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</figure>
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Waldo, G.S. et al. (January 2006). Engineering and characterization of a superfolder green fluorescent protein. Retrieved from http://www.nature.com/naturebiotechnology
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</p>
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<figcaption>
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Figure 4. Surface coating analysis of recombinant proteins on hydrophilic glass slides (left) and hydrophobic polystyrene (PS) plates (right).
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</figcaption>
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Latest revision as of 15:13, 21 October 2019


T7 promoter+csgA-linker-mfp5-linker-sfGFP

This composite part is meant to express csgA-linker-mfp5-linker-sfGFP fusion genes under T7 promoter. CsgA is an amyloid-like protein encoded on genome of E.coli MG1655 providing mechanical cohesive strength. Mfp5 is mussel foot proteins from Mytilus galloprovincialis responsible for interface adhesion. Compared to T7 promoter+csga-linker-mfp5-linker-His( BBa_K3089021), we have added sfGFP to characterize the expression of the recombinant protein. It is a robustly folded version of GFP, called ‘superfolder’ GFP, that folds well even when fused to poorly folded polypeptides (Waldo et al, 2006).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 47
    Illegal PstI site found at 400
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 400
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 585
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
    Illegal PstI site found at 400
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
    Illegal PstI site found at 400
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 780


Characterization

The circuit of the protein BBa_K30889034

Fluorescence analysis

T7 promoter+csgA-linker-mfp5-linker-sfGFP was cloned into pET28b and transformed into E.coli BL21 (DE3).We grew 25-ml cultures of E. coli BL21 (DE3) bearing csgA-linker-mfp5-linker-sfGFP in LB medium containing kanamycin (50 mg/ml) overnight. We grew 1000-fold dilutions in 200-μL cultures to ~0.2/0.5/0.8 OD600 nm in a 96-well plate with cover and induced them at 37℃ with 500μM IPTG for 22 h. OD600nm and fluorescence were measured (488-nm excitation, 530-nm emission,10-nm bandpass for GFP) with a Microplate Fluorescence Reader (THERMO Varioskan Flash). Fluorescence was normalised by dividing by the OD600 nm. We continuously monitored the OD600nm and fluorescence of these four strains and plotted the graph for their growth and induced fluorescence. We added IPTG to these strains at different times of their log phase, such as OD600nm=0.2（early）, 0.5（medium）, 0.8（late）.

Results were measured by the ratio of fluorescence to OD600nm. CsgA-Mfp5-sfGFP had a relatively much poorer expression compare with sfGFP, which was used as control (Figure 1ABC).

Figure 2. Normalised fluorescence by dividing with OD600nm of sfGFP fused constructs（A-C）、OD600m(D-F) and fluorescence(G-I). Arrows indicate timing to add IPTG. Six repeats were monitored for each group and anomalies below 0 was ignored.
Figure 3. Normalized fluorescence (Fluorescence/OD600nm) measurement of CsgA-linker-Mfp5-linker-sfGFP. 500uM IPTG was added into cultures when it reached OD600nm at 0.2, 0.5, 0.8 separately after a 1000-fold dilution from overnight cultures. Arrows indicate timing to add IPTG. A.U. (arbitrary units) calculated by dividing fluorescence with OD600nm value. Six repeats were monitored for each group and anomalies below 0 was ignored.
Figure 4. Fluorescence curve of CsgA-linker-Mfp5-linker-sfGFP

Interestingly, adding inducer in early stage of log phase (OD=0.2) would delay the growth of CsgA-linker-Mfp5-linker-sfGFP (Figure 1D), letting them enter to lag phase again. During this second-lag-phase, the fluorescence was growing continuously (Figure 2G), which means proteins was still accumulating in cells. However, when growth entered log phase again, the normalised fluorescence reduced to some extent due to the rapid increase of OD600nm value. These phenomena are generally not observed when adding IPTG in medium (OD=0.5) and late log phase(OD=0.8)( Figure 1EF). For what we concern is the total yield, in other words, the absolute fluorescence of the culture. Results showed adding IPTG in early log phase significantly reduced overall expression, and the fluorescence reached its peak in 5 hours (Figure 4).

We concluded that we should add inducer in late log phase for higher-level expression and 5 hours’ induction was enough to get a highest yield.

Reference

Waldo, G.S. et al. (January 2006). Engineering and characterization of a superfolder green fluorescent protein. Retrieved from http://www.nature.com/naturebiotechnology