Difference between revisions of "Part:BBa K3075003"
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=== Introduction ===
 
=== Introduction ===
  
−
LXYL-p1-2-SpyTag-His consists of the enzyme Beta-D-xylosidase fused to a C-terminal short polypeptide tag (Spytag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The additional SpyTag enables the enzyme to be attached to the Assemblase scaffold designed by the 2018 UNSW iGEM team via a Spy-Catcher-Tag conjugation system. This bioconjugation system is not limited to the Assemblase scaffold, and can be utilised for the bioconjugation of LXYL-p1-2 to any recombinant protein containing the SpyCatcher.
+
LXYL-p1-2-SpyT consists of the enzyme Beta-D-xylosidase fused to a C-terminal short polypeptide tag (Spytag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The additional SpyTag enables the enzyme to be attached to the Assemblase scaffold designed by the 2018 UNSW iGEM team via a Spy-Catcher-Tag conjugation system. =
  
 
[[File:Lxyl_re.png]]
 
[[File:Lxyl_re.png]]
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=== Usage and Biology ===
 
=== Usage and Biology ===
  
−
In Lentinula edodes, Beta-D-xylosidase (LXYL) catalyses the hydrolysis of O-glycosyl bonds. The native function of LXYL was to catalyse the reaction of 7-beta-xylosyl-10-deacetylbaccatin III (XDB) to 10-deacetylbaccatin III (DB). The LXYL-p1-2 protein has been optimised to release the β-xylosyl group of 7-β-xylosyltaxanes (3). Specifically, the beta-hydrolase activity has been exploited to catalyse the removal of the xylose group from 7-beta-xylosyl-10-deacetyltaxol (XDT) to produce the intermediate 10-deacetyltaxol (DT) (3) (Figure 2). Recombinant LXYL-P1-2 has a sequence of 803 amino acid residues with a molecular mass of 85,975 Da.  
+
In Lentinula edodes, Beta-D-xylosidase (LXYL) catalyses the hydrolysis of O-glycosyl bonds. The native function of LXYL was to catalyse the reaction of 7-beta-xylosyl-10-deacetylbaccatin III (XDB) to 10-deacetylbaccatin III (DB). The LXYL-p1-2 mutant has been optimised to release the β-xylosyl group of 7-β-xylosyltaxanes (3). Specifically, the beta-hydrolase activity has been exploited to catalyse the removal of the xylose group from 7-beta-xylosyl-10-deacetyltaxol (XDT) to produce the intermediate 10-deacetyltaxol (DT) (3) (Figure 2). Recombinant LXYL-P1-2 has a sequence of 803 amino acid residues with a molecular mass of 85,975 Da.  
  
 
[[File:Lxyl_reaction2.png]]
 
[[File:Lxyl_reaction2.png]]
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=== Characterisation ===
 
=== Characterisation ===
  
−
pET19b vector provided by Dr Dominic Glover and linearised by PCR amplification. Linear gene fragments were purchased from Integrated DNA technologies (IDT). The gene constructs were assembled into the pET19b expression vector at the multiple cloning site via Gibson assembly with a 3-fold excess of insert. Gibson products were transformed into high efficiency T7 Express E. coli by heat shocking at 42°C and plated on ampicillin supplemented agar plates for selection. This resulted in seven (LXYL) transformant colonies, compared to zero colonies on the linear pET19b transformant negative control. Three colonies of  LXYL transformants were screened by colony PCR, where LXYL colonies revealed bands of an estimated size 1000 bp, which is below the expected size of 2528 bp, showing the colonies did not contain the desired LXYL gene (Figure omitted).
+
pET19b vector provided by Dr Dominic Glover was linearised by PCR amplification. Linear gene fragments were purchased from Integrated DNA technologies (IDT). The gene constructs were assembled into the pET19b expression vector at the multiple cloning site via Gibson assembly with a 3-fold excess of insert. Gibson products were transformed into high efficiency T7 Express E. coli by heat shocking at 42°C and plated on ampicillin supplemented agar plates for selection. This resulted in seven (LXYL) transformant colonies, compared to zero colonies on the linear pET19b transformant negative control. Three colonies of  LXYL transformants were screened by colony PCR, where LXYL colonies revealed bands of an estimated size 1000 bp, which is below the expected size of 2528 bp, showing the colonies did not contain the desired LXYL gene (Figure omitted).
  
 
Assembly of LXYL was re-attempted, by increasing the insert to vector ratio to 5:1 and screening more colonies by colony PCR. Attempts did not succeed.
 
Assembly of LXYL was re-attempted, by increasing the insert to vector ratio to 5:1 and screening more colonies by colony PCR. Attempts did not succeed.
  
 
As LXYL is a larger gene fragment (2.5kb), ligation into pET19b (5kb) would require a 2X excess of LXYL, instead of the 3X excess. However, excessive amounts of insert would unlikely to be the cause for non-assembly. It may be of benefit to increase the Gibson assembly incubation time to ensure there is sufficient time for the complementary overhangs to properly anneal between the insert and vector.
 
As LXYL is a larger gene fragment (2.5kb), ligation into pET19b (5kb) would require a 2X excess of LXYL, instead of the 3X excess. However, excessive amounts of insert would unlikely to be the cause for non-assembly. It may be of benefit to increase the Gibson assembly incubation time to ensure there is sufficient time for the complementary overhangs to properly anneal between the insert and vector.

Revision as of 00:09, 22 October 2019

LXYL-P1-2- SpyT-His


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1062
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
    Illegal NgoMIV site found at 168
  • 1000
    COMPATIBLE WITH RFC[1000]


Introduction

LXYL-p1-2-SpyT consists of the enzyme Beta-D-xylosidase fused to a C-terminal short polypeptide tag (Spytag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The additional SpyTag enables the enzyme to be attached to the Assemblase scaffold designed by the 2018 UNSW iGEM team via a Spy-Catcher-Tag conjugation system. =

Lxyl re.png

Figure 1: A visualisation of the attachment of LXYL-p1-2-SpyT (green) fusion protein to the Assemblase scaffold (red) via the Spy-Catcher-Tag system. Graphic produced by Linda Chen 2019.

The Hexahistidine tag is a common additive due to its high affinity for metal ions used in the purification technique of immobilized metal affinity chromatography (IMAC). Ni2+ ions were used for his-tag purification due to its high yield.

Usage and Biology

In Lentinula edodes, Beta-D-xylosidase (LXYL) catalyses the hydrolysis of O-glycosyl bonds. The native function of LXYL was to catalyse the reaction of 7-beta-xylosyl-10-deacetylbaccatin III (XDB) to 10-deacetylbaccatin III (DB). The LXYL-p1-2 mutant has been optimised to release the β-xylosyl group of 7-β-xylosyltaxanes (3). Specifically, the beta-hydrolase activity has been exploited to catalyse the removal of the xylose group from 7-beta-xylosyl-10-deacetyltaxol (XDT) to produce the intermediate 10-deacetyltaxol (DT) (3) (Figure 2). Recombinant LXYL-P1-2 has a sequence of 803 amino acid residues with a molecular mass of 85,975 Da.

Lxyl reaction2.png

Figure 2: Figure 2: 7-beta-xylosidase catalyses the hydrolysis of 7-beta-xylosyl-10-deacetyltaxol (XDT) to 10-deacetyltaxol (DT). Figure obtained from Ping Zhu (2017). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5454391/ (reference 2).

Characterisation

pET19b vector provided by Dr Dominic Glover was linearised by PCR amplification. Linear gene fragments were purchased from Integrated DNA technologies (IDT). The gene constructs were assembled into the pET19b expression vector at the multiple cloning site via Gibson assembly with a 3-fold excess of insert. Gibson products were transformed into high efficiency T7 Express E. coli by heat shocking at 42°C and plated on ampicillin supplemented agar plates for selection. This resulted in seven (LXYL) transformant colonies, compared to zero colonies on the linear pET19b transformant negative control. Three colonies of LXYL transformants were screened by colony PCR, where LXYL colonies revealed bands of an estimated size 1000 bp, which is below the expected size of 2528 bp, showing the colonies did not contain the desired LXYL gene (Figure omitted).

Assembly of LXYL was re-attempted, by increasing the insert to vector ratio to 5:1 and screening more colonies by colony PCR. Attempts did not succeed.

As LXYL is a larger gene fragment (2.5kb), ligation into pET19b (5kb) would require a 2X excess of LXYL, instead of the 3X excess. However, excessive amounts of insert would unlikely to be the cause for non-assembly. It may be of benefit to increase the Gibson assembly incubation time to ensure there is sufficient time for the complementary overhangs to properly anneal between the insert and vector.