Difference between revisions of "Part:BBa K3739081"
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===Characterization===
 
===Characterization===
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In order to eliminate threat from ''Phaeocystis globosa'' to the nuclear power plant cooling system, we design a genetic circuit which could express protein aim at this alga.  The cellulose binding module (CBM) and (histidine ammonia-lyase) HutH from ''Pseudomonas Putida'' were fused to protein CBM-HutH. The fusion protein could bind to the cellulose on the surface of ''P. globosa'' and catalyzes ''L''-histidine, secreted by ''P. globosa'' to ''trans''-urocanate. which then elevates the ROS around the ''P. globosa'' to damage the algal cells.</br>  
+
In order to eliminate threat from ''Phaeocystis globosa'' to the nuclear power plant cooling system, we design a genetic circuit which could express protein aim at this alga.  The cellulose binding module (CBM) and (histidine ammonia-lyase) HutH from ''Pseudomonas Putida'' were fused to protein CBM-HutH. The fusion protein could bind to the cellulose on the surface of ''P. globosa'' and catalyzes ''L''-histidine, secreted by ''P. globosa'' to ''trans''-urocanate. which then elevates the ROS around the ''P. globosa'' to damage the algal cells.<br/>  
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Promoter (BBa_K525998), RBS (BBa_B0030), his-CBM-HutH gene (BBa_K3739071), and terminator (BBa_B0010) were assembled into pET-28a(+) plasmid backbone to express the his-CBM-HutH. The constructed plasmid was transformed into ''Vibrio natriegens'' through electroporation. Positive colonies were selected by kanamycin preliminarily and then verified by colony 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 CBM 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'''). Because CBM own the ability of plastic binding activity, tube and beaker used after ultrasonication are made from glass or Teflon.</br>  
+
Promoter (BBa_K525998), RBS (BBa_B0030), his-CBM-HutH gene (BBa_K3739071), and terminator (BBa_B0010) were assembled into pET-28a(+) plasmid backbone to express the his-CBM-HutH. The constructed plasmid was transformed into ''Vibrio natriegens'' through electroporation. Positive colonies were selected by kanamycin preliminarily and then verified by colony 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 CBM 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'''). Because CBM own the ability of plastic binding activity, tube and beaker used after ultrasonication are made from glass or Teflon.<br/>  
−
The absorbance of ''trans''-urocanate was measured at 277 nm to obtain the data of concentration. Same concentration and volume of HutH and CBM-HutH solutions were added to the ''L''-histidine solution, and the value OD<sub>277</sub> was monitored and recorded. As shown in '''Fig. 3''', the OD<sub>277</sub> in two group was rising continuously, suggesting that CBM-HutH has the ability to catalyzes ''L''-histidine to ''trans''-urocanate.</br>
+
The absorbance of ''trans''-urocanate was measured at 277 nm to obtain the data of concentration. Same concentration and volume of HutH and CBM-HutH solutions were added to the ''L''-histidine solution, and the value OD<sub>277</sub> was monitored and recorded. As shown in '''Fig. 3''', the OD<sub>277</sub> in two group was rising continuously, suggesting that CBM-HutH has the ability to catalyzes ''L''-histidine to ''trans''-urocanate.<br/>
−
'''Fig. 1'''. The result of colony PCR. Plasmid pET-28a (+).</br>
+
'''Fig. 1'''. The result of colony PCR. Plasmid pET-28a (+).<br/>
−
'''Fig. 2'''. SDS-PAGE analysis of protein his-CBM-HutH. Target bands can be seen at about 69 kDa.</br>
+
'''Fig. 2'''. SDS-PAGE analysis of protein his-CBM-HutH. Target bands can be seen at about 69 kDa.<br/>
 
'''Fig. 3'''. Time course of the value of OD277.<br/>
 
'''Fig. 3'''. Time course of the value of OD277.<br/>
  

Revision as of 17:56, 21 October 2021


his-CBM-hutH

Histidine ammonia-lyase with a his-tag is involved in catalyzing the reaction of  changing L-histidine into trans-urocanate, CBM can anchor onto cellulose.


Usage and Biology

Characterization

In order to eliminate threat from Phaeocystis globosa to the nuclear power plant cooling system, we design a genetic circuit which could express protein aim at this alga. The cellulose binding module (CBM) and (histidine ammonia-lyase) HutH from Pseudomonas Putida were fused to protein CBM-HutH. The fusion protein could bind to the cellulose on the surface of P. globosa and catalyzes L-histidine, secreted by P. globosa to trans-urocanate. which then elevates the ROS around the P. globosa to damage the algal cells.
Promoter (BBa_K525998), RBS (BBa_B0030), his-CBM-HutH gene (BBa_K3739071), and terminator (BBa_B0010) were assembled into pET-28a(+) plasmid backbone to express the his-CBM-HutH. The constructed plasmid was transformed into Vibrio natriegens through electroporation. Positive colonies were selected by kanamycin preliminarily and then verified by colony 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 CBM 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). Because CBM own the ability of plastic binding activity, tube and beaker used after ultrasonication are made from glass or Teflon.
The absorbance of trans-urocanate was measured at 277 nm to obtain the data of concentration. Same concentration and volume of HutH and CBM-HutH solutions were added to the L-histidine solution, and the value OD277 was monitored and recorded. As shown in Fig. 3, the OD277 in two group was rising continuously, suggesting that CBM-HutH has the ability to catalyzes L-histidine to trans-urocanate.
Fig. 1. The result of colony PCR. Plasmid pET-28a (+).
Fig. 2. SDS-PAGE analysis of protein his-CBM-HutH. Target bands can be seen at about 69 kDa.
Fig. 3. Time course of the value of OD277.

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 537
    Illegal NgoMIV site found at 973
    Illegal NgoMIV site found at 1708
    Illegal NgoMIV site found at 1944
    Illegal AgeI site found at 283
    Illegal AgeI site found at 373
    Illegal AgeI site found at 610
    Illegal AgeI site found at 1437
    Illegal AgeI site found at 1933
  • 1000
    COMPATIBLE WITH RFC[1000]