Difference between revisions of "Part:BBa K3739086"
 
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===Usage and Biology===
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===Biology===
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FlAsH system was previously reported as a fluorescence detector for secreted proteins<sup>1</sup>. The protein labeled of tetracysteine motif tag (FlAsH tag, FT in short) can react with biarsenical compound FlAsH, showing great fluorescence at 528 nm emission. FlAsH system is used to quantify and qualify the secretion efficiency. We used his-hutH-FT to verify the secretion effect.
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===Usage===
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We ligased the induced promoter+RBS (<partinfo>BBa_K525998</partinfo>) and the parts (<partinfo>BBa_K3739086</partinfo>) on the expression vector pSB1C3 by standard assembly (<partinfo>BBa_K3739087</partinfo>). Then the ligation mixture was transformed into ''E.coli'' BL21 (DE3), and the correct recombinant one was confirmed by chloramphenicol, colony PCR and sequencing.
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===Characterization===
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1.Agarose Gel Electrophoresis<br/>
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After <partinfo>BBa_K3739087</partinfo> was constructed on vector pSB1C3 and transformed into ''E. coli'' BL21 (DE3), colony PCR was done to verify that the plasmid was correct. Data is shown below:<br/>
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[[File:T--XMU-China--86-1.png|300px]]<br/>
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'''Fig. 1.''' Colony PCR results of BBa_K3739087<br/>
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2. Qualification of LMT secretion efficiency<br/>
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After cultivation for 5 hours at 37℃ and induction for 2 hours with 0.1mM IPTG, the cell supernatant was separated by centrifugation. The supernatant along with 2 μM FlAsH-EDT2 and 1 mM DTT were added to wells in a 96-well plate. Following incubation in the dark for 1 h at 37 °C, fluorescence was measured by 503 nm excitation and 528 nm emission, and each value was normalized by OD<sub>600</sub>.<br/>
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[[File:T--XMU-China--86-2.png|300px]]<br/>
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'''Fig. 2.''' Fluorescence intensity/OD<sub>600</sub> of cell supernatant after incubation in the dark for 1h.<br/>
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The result shows that the fluorescence intensity/OD<sub>600</sub> of the group Aly01 and LMT is significantly higher than native control group, which proves that these 2 signal peptides can function well.
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===Reference===
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1. Haitjema, C. H.; Boock, J. T.; Natarajan, A.; Dominguez, M. A.; Gardner, J. G.; Keating, D. H.; Withers, S. T.; DeLisa, M. P., Universal Genetic Assay for Engineering Extracellular Protein Expression. ''ACS Synthetic Biology'' '''2014''', 3 (2), 74-82.
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===Characterization===
 
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The ''hutH'' gene from ''Pseudomonas putida'' was heterologously expressed in chassis bacteria to produce histidine ammonia-lyase (HutH), which could catalyze ''L''-histidine to ''trans''-urocanate. Promoter (BBa_J23100), RBS (BBa_B0030), ''hutH'' gene, and terminator (BBa_B0010) were assembled into pET-28a(+) plasmid backbone to express the HutH.<br/>
 
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The constructed plasmid was transformed into ''Vibrio natriegens'' through electroporation. Positive colonies were selected by kanamycin preliminarily and then verified by regular PCR ('''Fig. 1''') and sequencing. Then, the colony with the corrected sequence was cultivated to express HutH target protein. After ultrasonication broke and centrifugation, GE AKTA Prime Plus FPLC System was employed to purify the HutH from the supernatant. Purified protein was verified by electrophoresed on a sodium dodecyl sulfate (SDS)-10% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining ('''Fig. 2''').<br/>
 
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The absorbance of ''L''-histidine was measured at 277 nm (ε = 18000 (mol · L-1)-1 · cm-1) to obtain the data of concentration, which was used to calculate the kinetic constants of Km and ''kcat'' ('''Fig. 3'''). <br/>
 
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Thus, these data demonstrate that HutH works well to catalyze ''L''-histidine to ''trans''-urocanate.<br/>
 
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'''Fig. 1'''. The result of regular PCR. Plasmid pET-28a(+).<br/>
 
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'''Fig. 2'''. SDS-PAGE analysis of protein in lysate of ''Vibrio natriegens'' and the eluant. Target bands (55.7 kDa) can be observed at the position around 50 kDa.<br/>
 
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'''Fig. 3'''. The relationship of 1/Enzyme activity and 1/concentration of ''L''-histidine.
 
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 23:04, 21 October 2021


his-hutH-FT

Histidine ammonia-lyase with a his-tag is involved in catalyzing the reaction of changing L-histidine into trans-urocanate. The proteins labeled of tetracysteine motif tag (FlAsH tag, FT in short) can be detected with the biarsenical compound FlAsH.


Biology

FlAsH system was previously reported as a fluorescence detector for secreted proteins1. The protein labeled of tetracysteine motif tag (FlAsH tag, FT in short) can react with biarsenical compound FlAsH, showing great fluorescence at 528 nm emission. FlAsH system is used to quantify and qualify the secretion efficiency. We used his-hutH-FT to verify the secretion effect.

Usage

We ligased the induced promoter+RBS (BBa_K525998) and the parts (BBa_K3739086) on the expression vector pSB1C3 by standard assembly (BBa_K3739087). Then the ligation mixture was transformed into E.coli BL21 (DE3), and the correct recombinant one was confirmed by chloramphenicol, colony PCR and sequencing.

Characterization

1.Agarose Gel Electrophoresis
After BBa_K3739087 was constructed on vector pSB1C3 and transformed into E. coli BL21 (DE3), colony PCR was done to verify that the plasmid was correct. Data is shown below:
T--XMU-China--86-1.png
Fig. 1. Colony PCR results of BBa_K3739087
2. Qualification of LMT secretion efficiency
After cultivation for 5 hours at 37℃ and induction for 2 hours with 0.1mM IPTG, the cell supernatant was separated by centrifugation. The supernatant along with 2 μM FlAsH-EDT2 and 1 mM DTT were added to wells in a 96-well plate. Following incubation in the dark for 1 h at 37 °C, fluorescence was measured by 503 nm excitation and 528 nm emission, and each value was normalized by OD600.
T--XMU-China--86-2.png
Fig. 2. Fluorescence intensity/OD600 of cell supernatant after incubation in the dark for 1h.

The result shows that the fluorescence intensity/OD600 of the group Aly01 and LMT is significantly higher than native control group, which proves that these 2 signal peptides can function well.

Reference

1. Haitjema, C. H.; Boock, J. T.; Natarajan, A.; Dominguez, M. A.; Gardner, J. G.; Keating, D. H.; Withers, S. T.; DeLisa, M. P., Universal Genetic Assay for Engineering Extracellular Protein Expression. ACS Synthetic Biology 2014, 3 (2), 74-82.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 162
    Illegal NgoMIV site found at 598
    Illegal NgoMIV site found at 1333
    Illegal NgoMIV site found at 1569
    Illegal AgeI site found at 235
    Illegal AgeI site found at 1062
    Illegal AgeI site found at 1558
  • 1000
    COMPATIBLE WITH RFC[1000]