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:



EcoRI     NotI                 XbaI                        SpeI            NotI               PstI

GAATTCGCGGCCGC      TˇCTAGA     insert    AˇCTAGT     GCGGCCGCCTGCAG   altered BioBrick

This standard is fully compatible with "traditional BB" (Knight et al.) and only leaves different "scar" after ligation.

                                                                                      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.

Slovenian Team Jewelbox
Following parts were deposited into BioBrick database:

BBa_K133000 CF-mCherry-RGD-Hisstop2299
BBa_K133001 CF-multiHP-RGD-Histop2116
BBa_K133002 CF-RGD_Histop1583
BBa_K133003 CF-UB331708
BBa_K133004 CF-UB33-RGD-Histop1746
BBa_K133005 CF-UreB-RGD-Histop3296
BBa_K133006 CF213-multiHP1169
BBa_K133007 CF213-multiHP-CF215-RGD-Histop2122
BBa_K133008 CF213-UB33799
BBa_K133009 CF213-UB33-CF215-RGD-Histop1752
BBa_K133010 CF213636
BBa_K133011 CF213-UreB-linker2381
BBa_K133012 CF213-UreB-linker-CF215-RGD-Histop3334
BBa_K133013 CF215-RGD-Histop947
BBa_K133015 CF215-mCherry-RGD-Histop1663
BBa_K133016 CF1545
BBa_K133017 mCherry-RGD-Histop746
BBa_K133018 CMV-CF-UreB-RGD-Histop3956
BBa_K133019 CMV-CF213-multiHP-CF215-RGD-Histop2782
BBa_K133020 CMV-CF213-multiHP-CF215-RGD-Histop (term.)2782
BBa_K133021 CMV-ss-CF-RGD-Histop2324
BBa_K133022 CMV-ss-CF213-multiHP-CF215-RGD-Histop2863
BBa_K133023 CMV-ss-CF213-multiHP-CF215-RGD-Histop (term)2863
BBa_K133024 CMV-ss-CF-UreB-RGD-Histop4037
BBa_K133027 HpaA-UreB-RGD-Histop2531
BBa_K133028 HpaA-GFP-RGD-Histop1541
BBa_K133029 HpaA-multiHP-RGD-Histop1351
BBa_K133031 HpaA-CF-RGD-Histop2369
BBa_K133032 HpaA-multiHP1313
BBa_K133034 LPP-CF-RGD-Histop1667
BBa_K133035 Met-His 21
BBa_K133036 RGD-Histop30
BBa_K133037 T7-CF213-UB33-CF215-RGD-Histop1752
BBa_K133038 T7-CF-RGD-Histop1583
BBa_K133039 T7-CF-UREB-RGD-Histop3296
BBa_K133040 T7-UreB-Histop3296
BBa_K133041 T7 promotor3273
BBa_K133042 TetR(RBS)-GFP761
BBa_K133043 tetR-RBS-CF-RGD-Histop1624
BBa_K133044 TetR(RBS)35
BBa_K133045 TetR-RBS-CF213-UB33-CF215-RGD-Histop1793
BBa_K133046 CF215909
BBa_K133047 TetR-GFP780
BBa_K133048 TetR-UreB-Histop1796
BBa_K133049 TetR-CF-UreB-RGD-Histop1805
BBa_K133050 TetR-CF-RGD-Histop1643
BBa_K133051 TetR-CF-mCherry-RGD-Histop 
BBa_K133053 UreB-linker1739
BBa_K133054 UreB-RGD-Histop1745
BBa_K133055 mCherry708
BBa_K133056 HpaA-LPP-RGD-Histop902
BBa_K133057 HpaA780
BBa_K133058 CF-multiHP2078
BBa_K133059 RGD9
BBa_K133060 (TMTIR)TLR9645
BBa_K133061 CD4ecto1113
BBa_K133062 gyrHP660
BBa_K133063 (TIR)TLR3453
BBa_K133064 (TIR)TLR9585
BBa_K133065 (TMTIR)TLR3600
BBa_K133066 GFP717
BBa_K133067 (TMTIR)TLR4621
BBa_K133068 CD4sh-(TIR)TLR31647
BBa_K133069 (TMTIR)TLR3stop603
BBa_K133070 gyrEC660
BBa_K133071 UreB1707
BBa_K133072 CMV-SS-gyrHP1401
BBa_K133073 CMV-SS-gyrHP-linker1433
BBa_K133074 CMV-SS-gyrHP-2xlinker1465
BBa_K133075 CMV-SS-gyrEC1401
BBa_K133076 CMV-SS-gyrEC-linker1433
BBa_K133077 CMV-SS-gyrEC-2xlinker1465
BBa_K133078 CMV-SS-HA-UreB-gyrHP3150
BBa_K133079 CMV-SS-HA-UreB-gyrHP-linker3182
BBa_K133080 CMV-SS-HA-UreB-gyrEC3150
BBa_K133081 CMV-SS-HA-UreB-gyrEC-linker3182
BBa_K133082 CMV-SS-HA-UreB-gyrEC-2xlinker3214
BBa_K133083 CMV-SS-HA-UreB-gyrEC-2xlinker-(TMTIR)TLR9-Histop3896
BBa_K133084 CMV-SS-gyrHP-2xlinker-(TMTIR)TLR9-Histop2147
BBa_K133085 CMV-SS-gyrEC-2xlinker-(TMTIR)TLR9-Histop2147
BBa_K133086 CMV-SS-gyrEC-linker-(TMTIR)TLR9-GFP-Histop2838
BBa_K133087 CMV-SS-gyrHP-linker-(TMTIR)TLR9-GFP-Histop2838
BBa_K133088 CMV-SS-gyrHP-(TMTIR)TLR92054
BBa_K133089 CMV-SS-HA-ureB-GpA2561
BBa_K133090 CMV-SS-HA-CD4ecto1892
BBa_K133091 CMV-SS-HA-CD4ecto-(TMTIR)TLR9-Histop2574
BBa_K133092 CMV-SS-HA-CD4ecto-(TMTIR)TLR3stop2503
BBa_K133093 CMV-SS-HA-UreB-CD4ecto3605
BBa_K133094 CMV-SS-HA-UreB-CD4ecto-(TMTIR)TLR9-Histop4287
BBa_K133095 CMV-SS-HA-multiHP-CD4ecto-(TMTIR)TLR3-GFP-Histop3785
BBa_K133096 CMV-SS-HA-multiHP-CD4ecto-(TMTIR)TLR9-Histop3107
BBa_K133097 CMV-SS-HA-multiHP-CD4ecto2425
BBa_K133098 CMV-SS-HA-(TMTIR)TLR41398
BBa_K133099 CMV-SS-HA-CD4sh-(TIR)TLR3-Histop2455
BBa_K133100 CMV-SS-HA-CD4sh-(TIR)TLR3-(TIR)TLR9-Histop3048
BBa_K133101 CMV-SS-HA-multiHP-CD4sh-(TIR)TLR3-Histop2988
BBa_K133102 CMV-SS-HA-UreB-CD4sh-(TIR)TLR3-(TIR)TLR9-Histop4761
BBa_K133103 CMV-SS-HA-(TMTIR)TLR4-Histop1427
BBa_K133104 CMV-SS-HA-(TMTIR)TLR4-GFP-Histop2150
BBa_K133105 (TIR)TLR9-GFP-Histop1337
BBa_K133106 (TMTIR)TLR9-GFP-Histop1397
BBa_K133107 (TIR)TLR9-Histop614
BBa_K133108 (TMTIR)TLR9-Histop674
BBa_K133109 (TMTIR)TLR3-GFP-Histop1352
BBa_K133110 CMV-SS-gyrEC-linker-(TMTIR)TLR92086
BBa_K133111 CMV-SS-HA-multiHP-(TMTIR)TLR41931
BBa_K133112 CMV-SS-HA-multiHP-(TMTIR)TLR4-Histop1960
BBa_K133113 CMV-SS-HA-multiHP-(TMTIR)TLR4-GFP-Histop2683
BBa_K133114 multiHP-Histop554
BBa_K133115 GFP-Histop746
BBa_K133116 UreB-Histop1736
BBa_K133117 CD4sh-(TIR)TLR3-(TIR)TLR9-Histop2269
BBa_K133118 CD4sh-(TIR)TLR3-Histop1676
BBa_K133119 CMV-SS-gyrHP-linker-(TMTIR)TLR92086
BBa_K133120 CMV-SS-multiHP-Histop1297
BBa_K133121 CMV-HA-UreB2403
BBa_K133122 CMV-SS-HA-UreB-(TMTIR)TLR43111
BBa_K133123 CMV-SS-HA-UreB-gyrEC-linker-(TMTIR)TLR93835
BBa_K133124 CMV-SS-HA-UreB-Histop2513
BBa_K133125 CMV-HA-UreB-Histop2432
BBa_K133126 CMV-SS-HA771
BBa_K133127 CMV-SS-HA-multiHP1304
BBa_K133128 CMV-SS-HA-UreB2484
BBa_K133129 CMV-SS-UreB2448
BBa_K133131 CMV-HA-UreB-GFP-Histop3155
BBa_K133132 linker24
BBa_K133133 Histop21
BBa_K133134 CMV-SS-HA-UreB-CD4ecto-(TMTIR)TLR3stop4216
BBa_K133135 CMV-SS-HA-UreB-CD4sh-(TIR)TLR3-Histop4168
BBa_K133136 CMV-SS-HA-UreB-gyrEC-linker-(TMTIR)TLR9-GFP-Histop4587
BBa_K133137 CMV-SS-HA-CD4ecto-(TMTIR)TLR3-GFP-Histop3252

We improved the following biobricks from the Registry.