Part:BBa_K1352000
Ag43 with PstI sites removed under the control of pBad/araC promoter
Abstract
We describe the creation of BioBrick BBa_K1352000, the modularized version of BBa_K759001, which is compatible with iGEM Assemly Standard RCF10. Previous work by the Aberdeen 2014 iGEM team had clearly shown that both Ag43 containing BioBricks BBa_K346007 and BBa_K759001 contained the 6 native PstI sites that characterise the native Ag43 sequence. Also, BBa_K542009 was shown to contain an additional XbaI site and to have a smaller size than predicted. They were thus found to be non-compliant with assembly Standard 10. We describe the design and construction of the new BioBrick BBa_K1352000, and its verification using restriction digestion and DNA sequencing. Finally, we show that antigen 43 protein expression of biobrick BBa_K1352010 was not affected by the PCR-based Site-Directed Mutagenesis process. The biobrick works biologically as intended after modification and has the same response to arabinose induction: formation of culture aggregation.
1.Structure and function
Antigen 43 (Ag43), the product of the flu gene, is a cell-surface autotransporter protein found in Escherichia coli. It is expressed at about 50, 000 copies/cell and is initially synthesised as a precursor of 1039 amino acids. Upon removal of the signal peptide, the protein is transported to the cell surface and is composed of an α subunit (499 amino acids) at the N-terminus and a β subunit (488 amino acids) at the C-terminus (Kj\aergaard et al., 2002). Ag43 is mainly known to induce cell-to-cell aggregation and be involved in biofilm formation. However, as the necessary information required for auto transportation resides in the protein itself (Kj\aergaard et al., 2002), the main of our project was to use it as a platform for displaying specific peptides on the surface of E. coli. To accomplish this aim, we have modularized and characterized Biobrick BBa_K759001.
Source: Westfälische Wilhelms-Universität Münster, I. (2014).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1945
Illegal NgoMIV site found at 3169
Illegal NgoMIV site found at 3679
Illegal NgoMIV site found at 3700
Illegal AgeI site found at 979
Illegal AgeI site found at 2695
Illegal AgeI site found at 3439
Illegal AgeI site found at 3853
Illegal AgeI site found at 4095 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
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