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Team:Slovenia/Results/Biobricks
From 2008.igem.org
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This year’s team Slovenia has highly surpassed the efforts of the previous Slovenian teams, creating and depositing no less than 132 new Biobricks and thus making a vital contribution to the expansion of the registry. Basic Biobricks have been sequenced.
About 20 BioBricks were shown to be functional or functional parts of larger assemblies.
In contrast to previous years, when we made most of the protein domain fusions by PCR ligation, we used Biobrick assembly using the standard restriction sites for ligation. In order to preserve the reading frame we used the following standard of Biobrick construction as in previous years:
GAATTCGCGGCCGC TTˇCTAGAG insert TAˇCTAGTA GCGGCCGCCTGCAG standard BioBrick EcoRI NotI XbaI SpeI NotI PstI GAATTCGCGGCCGC TˇCTAGA insert AˇCTAGT GCGGCCGCCTGCAG altered BioBrick
BioBricks are NOT in reading frame EcoRINotI TXbaIG insert 1 TACTAGAG insert 2 TSpeIA NotIPstI standard scar
EcoRINotI XbaI insert 1 ACTAGA insert 2 SpeI NotIPstI changed scar BioBricks are in reading frame
Improvement of Biobricks from previous years
Some of our work includes characterization and improvements of previously deposited bricks, for example an addition of a ribosome binding site to BBa_R0040 and removal of stop codons from BBa_J52642 and BBa_I712084 so that it allows their fusion with other proteins also at non-C-terminal sites. Most of our work presents bricks that combine H. pylori antigens with different bacterial and eukaryotic promoters, thus allowing for future novel combinations of these antigens with active immune response enhancers. Special features of selected Biobricks  Biobrick T7-CF-RGD-HISTOP (BBa_K133038) is composed of: T7 promotor with RBS; chimeric flagellin made of N-terminal of E. coli flagellin, variable region of H. pylori, C-terminal of E. coli flagellin; RGD sequence; His tag with stop codon. Our basic chimeric flagellin constuct is composed of the strong T7 promotor and RBS for high level of expression in E. coli strains. The reading frame consists of chimeric flagellin composed of N-terminal segment of E. coli FliC, central variable segment of H. pylori flagellin FlaA and C-terminal conserved segment of E. coli FliC, that activates both innate immune response through TLR5, while providing antigenic determinants of H. pylori FlaA. Additionally fusion protein contains a RGD segment for integrin attachment and internalization for improved use as a mucosal adjuvant, His tag for detection and stop codon with terminator. This construct causes that flagellin of H. pylori is detected by the immune system (TLR5).  Biobrick T7-CF-UREB-RGD-HISTOP (BBa_K133039) is composed of: T7 promotor with RBS; chimeric flagellin made of N-termini of E. coli flagellin, variable region of H. pylori, C-termini of E. coli flagellin; urease B antigen; RGD sequence; His tag with stop codon. This Biobrick is the extended version of the upper Biobrick with additional antigen H. pylori urease B. It therefore contains a central segment and urease B of H. pylori as target antigen. Functionality of this Biobrick was tested and it produced a high amount of active protein, which was purified and used in animal immunization, resulting in a high titer of antibodies.  Biobrick CMV-ss-CF213-MULTI-CF215-RGD-HIS-STOP (BBa_K133023) is composed of: CMV promotor with RBS; signal sequence for secretion outside the cell, chimeric flagellin - multiepitope (urease epitope, VacA epitope, HpaA epitope from H. pylori) made of N-termini of E. coli flagellin, variable region of H. pylori with inserted multiepitope, C-termini of E. coli flagellin; RGD sequence; His tag with stop codon. This Biobrick is intended for use as DNA vaccine for expression in human cells and secretion, which provides transactivation of cells (e.g. lamina propria dendritic cells) that express TLR5. As an additional epitope this construct contains synthetic multiepitope within the variable segment of flagellin. This construct was demonstrated to work and cause strong activation and production of cytokines.  Biobrick CMV-ss-HAmultiCD4eTMTIR3Histop (BBa_K133095) is composed of: CMV promotor with RBS; signal sequence for secretion outside the cell, HA tag, multiepitope, CD4 ectodomain for dimerization; transmembrane and TIR domain of TLR3; His tag with stop codon. This Biobrick is intended for use as mammalian cell/animal DNA vaccine for expression of membrane anchored protein, consisting of synthetic multiepitope antigen, CD4 dimerization ectodomain, transmembrane and cytosolic domain of TLR3 and His-tag for protein isolation. Proper localization is ensured by the signal sequence and transmembrane domain of TLR3, which targets the protein towards ER/endosomes, where the antigen is processed in the endosome.
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