Part:BBa_K3904319
pET-28a(+) with N-His tag and TEV recoginition 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-28a(+) with XbaI and NdeI and ligated it with RBS, spacer, His-tag, and TEV recognition sequence cassette. This way, we get plasmid with the N-His tag, which can be separated from the protein by TEV protease.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 5161
Illegal XbaI site found at 5030 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 5161
Illegal NheI site found at 5122
Illegal NotI site found at 5186 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 5161
Illegal BglII site found at 4964
Illegal BamHI site found at 5155
Illegal XhoI site found at 5195 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 5161
Illegal XbaI site found at 5030 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 5161
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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 2263
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