Difference between revisions of "Part:BBa K3075002"
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=== Introduction ===
 
=== Introduction ===
  
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DBAT-Snooptag-His consists of the enzyme 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT) fused to a short C-terminal polypeptide tag (Snooptag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The sequence of DBAT which was used, originated from ''Taxus cuspidata'' (Japanese yew), with a double mutation of G38R/F301V (2). The SnoopTag is a small polypeptide tag that spontaneously forms an isopeptide bond between reactive amino acid side chains to its corresponding SnoopCatcher (Brune, 2017). This system opens up a variety of applications, utilising the catcher-tag conjugation system for bioconjugation and synthetic assembly of the DBAT enzyme to SnoopCatcher containing proteins.
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TycA-SpyTag-His consists of the enzyme Tyrocidine synthase 1 fused at the C-terminus to a short polypeptide tag (Spytag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. Our team utilised a sequence originating from ''Brevibacillus parabrevis'' with a S563A amino acid mutation. (1) (2)
  
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[[File:Part-BBa_K3075001-Introduction.png]]
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[[File:TycA_Reaction.png]]
  
 
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.
 
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.
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=== Usage and Biology ===
 
=== Usage and Biology ===
  
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This protein naturally participates in the synthesis of baccatin III, where it catalyses the final acetylation of 10-deacetylbaccatin III. Baccatin III synthesis is a subpathway of paclitaxel biosynthesis, which is itself part of Alkaloid biosynthesis. The mutant however, has been designed to catalyse the acetylation of 10-deacetyltaxol (DT) with a catalytic efficiency approximately six times higher than that of the wild-type. (2) The recombinant mutant enzyme has a length of 440 amino acid residues, a molecular weight of 49,052 Da and an optimum pH of 7.5. (3)
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Tyrocidine Synthase 1 (TycA) is known to be involved in tyrocidine biosynthesis, a part of antibiotic biosynthesis. In the biosynthesis of the Paclitaxel side chain, TycA combines the beta-phenylalanine and the CoA thioester, and can also catalyse the production of phenylisoserinyl-CoA. (3) Recombinant TycA has a sequence of 1088 amino acid residues with a molecular mass of 122,672.
  
 
=== Characterisation ===
 
=== Characterisation ===
  
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The gBlock was assembled into the pET19b expression vector at the multiple cloning site via gibson assembly with a 3-fold excess of insert to vector <link to protocols>. Gibson products were transformed into high efficiency T7 Express E. coli (NEB) by heat shocking at 42°C and cells were plated on ampicillin supplemented agar plates for selection. Transformants were screened for recombinant plasmids by colony PCR (figure ??). Colonies resulting in amplicons with an observed molecular weight of approximately 1.5 kb were grown overnight in a 5 mL culture and plasmid DNA was extracted by miniprep and submitted for sequence confirmation via Sanger sequencing (Figure ??).  
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TycA gene inserts were obtained in two fragments (TycA-1 and TycA-2), with a 60 bp overlap between the two fragments. Ligation of both TycA-1 and TycA-2 fragments with pET-19b backbone (provided by Dr Dominic Glover) was attempted via Gibson Assembly. The assembly mix was incubated for 1 hour at 37°C before transforming into T7 Express E. coli cells by heatshocking at 42°C. Transformed cells were plated on an LB Agar plate supplemented with Ampicillin at working concentration for selection, and incubated overnight at 37°C. Transformant colonies were screened for recombinant plasmids by colony PCR under the following conditions: Initial denaturation at 97°C for 3 minutes, 30X cycles of: denaturation at 97°C for 10 seconds, annealing at 67.6°C for 30 seconds and extension at 72°C for 50 seconds. Final extension at 72°C for 5 minutes. PCR products were run on a 1% agarose gel in 1X TAE Buffer for 1 hour at 100 V. Colony PCR performed on the TycA colonies showed bands which did not align with the theoretical length of 3.383 kb. Sanger sequencing results confirmed that the colonies submitted did not contain the desired TycA gene insert (Figure omitted).
  
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Image
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As TycA is a larger gene fragment (3.3kb), we attempted to increase the incubation time during Gibson Assembly to allow for enough time for the complementary overhangs to properly anneal between the insert and vector.
  
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Figure 2. Recombinant DBAT-SnoopT-His gene amplified by colony PCR at annealing temperature 67.6°C and extension time 43 seconds, else as per protocol. 10 uL of PCR product was run on a 1% agarose gel at 100 V for 1 hour using 5 uL of 2-log DNA ladder (NEB) as a standard (Lane 1). Single band at ~1.5kb.
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The following conditions have been attempted:
 +
:*Gibson Assembly incubation – 15 minutes, 60 minutes.  
 +
:*Gibson master mix: commercial compared with home-made.
 +
:*Insert to vector: 3X excess, 5X excess.
  
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Figure 3. DBAT-SnoopT-His sequence chromatogram.
 
  
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'''Protein expression assay'''
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The 60 minutes (Figure 6A) incubation produced greater number of colonies compared to the 15 minutes (Figure 6B). This suggests the longer incubation time allows for the ligation of fragments into a functional circularised plasmid. Colony PCR results of these colonies revealed __________. The commercial master mix had more colonies than the home-made, but not significantly (Figure 6C).
  
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Cells containing a plasmid with the DBAT insert were grown up and a sample of this was used to perform a protein expression assay. Bug buster was used to separate soluble and insoluble proteins. LXYL was not successfully cloned thus could not be expressed.
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With multiple repetitions of the Gibson Assembly procedure, the lack of successful recombinant plasmids raises the question, that perhaps the gene fragments are not correctly synthesised. We plan to confirm the sequence of our gene fragments empirically, by Sanger sequencing.
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+
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Image
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Figure ?. Protein expression assay using bug buster to determine expression of 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT) as soluble and insoluble form.
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'''Purification'''
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Following the confirmation of protein expression indicated by bug buster gels, attempts were made to purify DBAT.
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Image
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Figure ?. SDS-PAGE of AKTA purification fractions of DBAT His-tagged protein
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'''Liquid Chromatography with tandem mass spectrometry'''
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Soluble protein bands (fractions 4-7) as well as a total protein lysate band at the same predicted molecular weight as DBAR were excised from the gel of purified fractions in Figure? And sent for analysis by Liquid Chromatography with tandem mass spectrometry (LCMSMS). This was performed to determine the identity of the protein bands by mapping peptides detected by LCMSMS onto the sequence of DBAT obtained from sequencing data of the cloned insert.
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Image
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Figure ?.  Liquid Chromatography with tandem mass spectrometry analysis of suspected DBAT protein bands excises form Figure ? protein gel. A: Total protein lysate sample. B: soluble protein sample taken from fractions 4-7.
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Revision as of 12:02, 21 October 2019

TycAS563A-SpyT-His


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2289
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2299
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1223
    Illegal BsaI.rc site found at 2143
    Illegal SapI.rc site found at 2499

Introduction

TycA-SpyTag-His consists of the enzyme Tyrocidine synthase 1 fused at the C-terminus to a short polypeptide tag (Spytag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. Our team utilised a sequence originating from Brevibacillus parabrevis with a S563A amino acid mutation. (1) (2)

TycA Reaction.png

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

Tyrocidine Synthase 1 (TycA) is known to be involved in tyrocidine biosynthesis, a part of antibiotic biosynthesis. In the biosynthesis of the Paclitaxel side chain, TycA combines the beta-phenylalanine and the CoA thioester, and can also catalyse the production of phenylisoserinyl-CoA. (3) Recombinant TycA has a sequence of 1088 amino acid residues with a molecular mass of 122,672.

Characterisation

TycA gene inserts were obtained in two fragments (TycA-1 and TycA-2), with a 60 bp overlap between the two fragments. Ligation of both TycA-1 and TycA-2 fragments with pET-19b backbone (provided by Dr Dominic Glover) was attempted via Gibson Assembly. The assembly mix was incubated for 1 hour at 37°C before transforming into T7 Express E. coli cells by heatshocking at 42°C. Transformed cells were plated on an LB Agar plate supplemented with Ampicillin at working concentration for selection, and incubated overnight at 37°C. Transformant colonies were screened for recombinant plasmids by colony PCR under the following conditions: Initial denaturation at 97°C for 3 minutes, 30X cycles of: denaturation at 97°C for 10 seconds, annealing at 67.6°C for 30 seconds and extension at 72°C for 50 seconds. Final extension at 72°C for 5 minutes. PCR products were run on a 1% agarose gel in 1X TAE Buffer for 1 hour at 100 V. Colony PCR performed on the TycA colonies showed bands which did not align with the theoretical length of 3.383 kb. Sanger sequencing results confirmed that the colonies submitted did not contain the desired TycA gene insert (Figure omitted).

As TycA is a larger gene fragment (3.3kb), we attempted to increase the incubation time during Gibson Assembly to allow for enough time for the complementary overhangs to properly anneal between the insert and vector.

The following conditions have been attempted:

  • Gibson Assembly incubation – 15 minutes, 60 minutes.
  • Gibson master mix: commercial compared with home-made.
  • Insert to vector: 3X excess, 5X excess.


The 60 minutes (Figure 6A) incubation produced greater number of colonies compared to the 15 minutes (Figure 6B). This suggests the longer incubation time allows for the ligation of fragments into a functional circularised plasmid. Colony PCR results of these colonies revealed __________. The commercial master mix had more colonies than the home-made, but not significantly (Figure 6C).

With multiple repetitions of the Gibson Assembly procedure, the lack of successful recombinant plasmids raises the question, that perhaps the gene fragments are not correctly synthesised. We plan to confirm the sequence of our gene fragments empirically, by Sanger sequencing.