Difference between revisions of "Part:BBa K3739086"
 
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===Biology===
 
===Biology===
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HutH exists in various microorganisms in nature that could catalyze ''L''-histidine to trans-urocanate ('''Fig. 1'''). In our project, the gene of HutH from ''Pseudomonas putida'' was expressed in chassis bacteria to produce histidine ammonia-lyase (HutH). After that, the product of trans-urocanate was further converted to glutamic acid.<br/>
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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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HutH is found in the livers of vertebrates and bacteria, such as ''E. coli'', ''Salmonella'' and ''pseudomonas'', which is specific to ''L''-histidine and can be inhibited by ''D''-histidine or imidazole.<br/>
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===Usage===
 
===Usage===
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His-tag (6×His) is expressed at the N end of HutH for protein purification. A strong promoter (<partinfo>BBa_J23100</partinfo>) and HutH parts (RBS-HutH-Terminator) were connected to the pET-28a(+) vector by the standard assembly, which was further transformed into ''E. coli'' DH5α. After being selected by kanamycin and verified by colony PCR and sequencing, the positive colony with corrected plasmid was obtained.  
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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.
 
===Characterization===
 
===Characterization===
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====1. Agarose gel electrophoresis (AGE)====
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1.Agarose Gel Electrophoresis<br/>
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When we were building this circuit, regular PCR was used to certify that the plasmid was correct. Target bands (1877 bp) can be observed at the position around 2000 bp ('''Fig. 2''').
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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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====2. SDS-PAGE====
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[[File:T--XMU-China--86-1.png|300px]]<br/>
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The plasmid verified by sequencing was successfully transformed into ''Vibrio natriegens''. After being cultivated and induced by ITPG, GE AKTA Prime Plus FPLC System was employed to get purified protein from broken supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in SDS-PAGE of HutH-his ('''Fig. 3'''), the target protein (55.7 kDa) could be observed at the position around 50 kDa on the purified protein lanes (FR), but not in the negative control groups (NC).
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'''Fig. 1.''' Colony PCR results of BBa_K3739087<br/>
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====3. Enzyme kinetic constants measurement====
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2. Qualification of LMT secretion efficiency<br/>
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The absorbance of ''L''-histidine was measured at 277 nm (ε = 18000 (mol · L<sup>-1</sup>)<sup>-1</sup> · cm<sup>-1</sup>) to obtain the data of concentration, which was used to calculate the kinetic constants of Km and ''kcat'' ('''Fig. 4''' and '''Table 1''')<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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'''Fig. 1'''. Schematic diagram of HutH catalytic process.<br/>
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[[File:T--XMU-China--86-2.png|300px]]<br/>
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'''Fig. 2'''. The result of regular PCR. Plasmid pET-28a (+).<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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'''Fig. 3'''. 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. 4'''. The relationship of 1/Enzyme activity and 1/concentration of ''L''-histidine.<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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'''Table 1'''. Enzyme kinetic constants with ''L''-histidine
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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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<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]