Difference between revisions of "Part:BBa K3739086"
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===Usage and Biology===
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===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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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===
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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.
 
===Characterization===
 
===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 (<partinfo>BBa_J23100</partinfo>), RBS (<partinfo>BBa_B0030</partinfo>), ''hutH'' gene, and terminator (<partinfo>BBa_B0010</partinfo>) were assembled into pET-28a(+) plasmid backbone to express the HutH.<br/>
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====1. Agarose gel electrophoresis (AGE)====
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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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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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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. 3'''). <br/>
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====2. SDS-PAGE====
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Thus, these data demonstrate that HutH works well to catalyze ''L''-histidine to ''trans''-urocanate.<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'''. The result of regular PCR. Plasmid pET-28a(+).<br/>
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====3. Enzyme kinetic constants measurement====
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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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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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'''Fig. 3'''. The relationship of 1/Enzyme activity and 1/concentration of ''L''-histidine.
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'''Fig. 1'''. Schematic diagram of HutH catalytic process.<br/>
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'''Fig. 2'''. The result of regular PCR. Plasmid pET-28a (+).<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/>
 +
'''Fig. 4'''. The relationship of 1/Enzyme activity and 1/concentration of ''L''-histidine.<br/>
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'''Table 1'''. Enzyme kinetic constants with ''L''-histidine
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 18:56, 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

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.
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.

Usage

His-tag (6×His) is expressed at the N end of HutH for protein purification. A strong promoter (BBa_J23100) 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.

Characterization

1. Agarose gel electrophoresis (AGE)

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').

2. SDS-PAGE

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).

3. Enzyme kinetic constants measurement

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. 4 and Table 1)
 Fig. 1. Schematic diagram of HutH catalytic process.
Fig. 2. The result of regular PCR. Plasmid pET-28a (+).
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.
Fig. 4. The relationship of 1/Enzyme activity and 1/concentration of L-histidine.
Table 1. Enzyme kinetic constants with L-histidine 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]