Part:BBa_K3904318

pET-28a(+) with N-His tag without any additional amino acids between tag and protein sequence

Introduction

Vilnius-Lithuania iGEM 2021 project AmeByelooks at amebiasis holistically and comprehensively, therefore target E. histolytica from several angles: prevention and diagnostics. Our team's preventive solution consists of probiotics engineered to produce naringenin - an antiprotozoal compound. Two strains of genetically modified microorganisms were chosen as the main chassis - world-renowned Lactobacillus casei BL23 (Lactobacillus paracasei) and Escherichia coli Nissle 1917. Furthermore, the team made specific gene deletions to enhance naringenin production and adapted a novel toxin-antitoxin system to prevent GMO spreads into the environment. The diagnostic part includes a rapid, point of care, user-friendly diagnostic test identifying extraintestinal amebiasis. The main components of this test are aptamers specific to the E. histolytica secreted proteins. These single-stranded DNA sequences fold into tertiary structures for particular fit with target proteins.

Usage and Biology

Regular pET-28a(+) has not been suitable for our experiments as it contained additional nucleotides coding random amino acids between His-tag and protein-encoding sequence. Protein without any additional amino acids is needed because it later has been used in SELEX experiments where ssDNA sequences specifically bind to protein because of specific protein structure features. Keeping this in mind, we restricted pET-28(a+) with XbaI and NdeI and ligated it with RBS, spacer, and His-tag cassette. This way, we get plasmid with the N-His tag.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 5140
  Illegal XbaI site found at 5030
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 5140
  Illegal NheI site found at 5101
  Illegal NotI site found at 5165
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 5140
  Illegal BglII site found at 4964
  Illegal BamHI site found at 5134
  Illegal XhoI site found at 5174
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 5140
  Illegal XbaI site found at 5030
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 5140
  Illegal XbaI site found at 5030
  Illegal NgoMIV site found at 137
  Illegal NgoMIV site found at 3184
  Illegal NgoMIV site found at 3344
  Illegal NgoMIV site found at 4932
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2263