Revision as of 14:08, 27 October 2010 by Kleinsorg (Talk | contribs)

miBricks- The Parts Concept

The parts we submit refer to the two core aims our project is focusing on: Reaching regulatory control (1) and specificity (2) of gene expression of any gene of interst in any target cell or tissue of choice. Therefor we engineered parts, that adress those two aims on two different regulatory levels. First, we engineered gene therapy vectors based on synthetic adeno associated viruses. On parts level, we provide about 50 plasmids that can be used for creating shuffeled AAV libraries or even rationally designed, recombinant AAV vectors. Those parts, we refer to as virobytes, are designed in a format, that is directly applicable for the Virobytes Assembly protocol we provide. On RNA level we provide the miTuner toolkit consisting of roughly 60 parts enabling gene expression control based on synthetic or cell-specific endogenous microRNAs. This toolkit consits of three main constructs: The pSMB_miMeasure binding site characterization standard and two pSMB_miTuner expression controlling plasmids. Furthermore, it contains 12 basic and 28 intermediate constrution parts, synthetic single microRNA binding sites as well as binding site patterns in BB-2 (RFC 12, Tom Knight) standard. This enables maximum flexibility for applications in many different contexts.

Main Measurement Constructs - Engineered

IDContentRegistry linkName
K3BGH(rc)/shRNA10(rc)/RSV(rc)/CMV/Luc2_sv40/CMV/Kozag_hRluc_BGHBBa_K337036 pSMB_miTuner Plasmid HD3
K4BGH(rc)/shRNA10(rc)/RSV(rc)/CMV_TetO2/Luc2_sv40/CMV/Kozag_hRluc_BGHBBa_K337038 pSMB_miTuner Plasmid HD4
miMsv40ter(rc)/eBFP(rc)/biCMV/eGFP(fw)/sv40ter(fw)BBa_K337049 pSMB_miMeasure

Synthetic Single Binding Sites

(Physical DNA not submitted)
DesignContentRegistry linkName
KD:97%perfect binding siteBBa_K337052shRNA miRhaat
KD:69%imperfect binding site: point mut 11BBa_K337053shRNA miRhaat
KD:28%imperfect binding site: bulge 16-18BBa_K337054shRNA miRhaat
KD:96%perfect binding siteBBa_K337055miR122
KD:64%imperfect binding site:BBa_K337056miR122
KD:24%imperfect binding site:BBa_K337057miR122

Endogenous Binding Site Patterns

IDContentRegistry link
1.3hsa-miR-122 Binding site pattern (3BS)BBa_K337000
1Ahsa-miR-122 Binding site pattern (4BS)BBa_K337003
1-5hsa-miR-122 Binding site pattern (2BS)BBa_K337004
3.7hsa-miR-122 Binding site pattern (2BS - additional 10bp Spacer)BBa_K337005
1.5hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12BBa_K337006
1.8hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12BBa_K337007
3.1hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12BBa_K337008
4.5hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12BBa_K337009
4.6hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12BBa_K337010
mir221-10L2hsa-miR-221 Binding site pattern (2BS)BBa_K337011

miTunig Kit - Basic Parts

IDContentRegistry linkComment
F1RSV fwBBa_K337012-
F2SV40 rcBBa_K337013-
F3RSV rcBBa_K337014-
F4BGH fwBBa_K337001-
F5BGH rcBBa_K337002 leads to insertion of BamHI site
F7microRNA 10HDBBa_K337016 template for creation of shRNA-like miRNA
F8CMV fwBBa_K337018-
F9FRT siteBBa_K337019-
F17Luc2BBa_K337030 compatible for insertion of microRNA binding sites

miTuning Kit - Intermediate Parts 1

IDContentRegistry linkPurpose of the cassette
F6luc2_sv40terBBa_K337015 Luc2 reporter gene with terminator referring to SV40 promoter
F15CMV_TetO2BBa_K337027 CMV promoter under control of Tet Operator for regulation of gene expression
R1Sv40(rc)/RSV(fw)BBa_K337045 bidirectional hybrid promoter
R5BGH(rc)/shRNA10(rc)BBa_K337051 synthetic microRNA template part
R13BGH(rc)/shRNA10(rc)/sv40(rc)/RSV(fw)BBa_K337020tuning construct cloning into reporter plasmid backbone containing a binding site against synthetic shRNA-like miRNA
R20BGH(rc)/shRNA6(rc)/RSV(rc)/CMV_TetO2(fw)BBa_K337031tuning construct core containing Tet Operator for On-Targeting together with repressor construct
R21Luc2(rc)_sv40(rc)/CMV(fw)BBa_K337033 bidirectional hybrid promoter with Luc2 reference gene under control of SV40 promoter
R32Kozag_hRluc/BGH(fw)BBa_K337037reference reporter
R33TetR_mut(XhoI/XbaI)BBa_K337039mutagenized TetR including cutting sites to paste miRNA binding sites
T1BGH(rc)_CMV/TetR/BGH(fw)/BGH(rc)BBa_K337041repressor construct for On-Targeting together with tuning construct

Tuning Kit - Intermediate Parts 2

IDContentRegistry linkName
K1BGH(rc)/shRNA10(rc)/sv40(rc)/RSV/Luc2_sv40/CMV/Kozag_hRluc_BGHBBa_K337032 pSMB_miTuner Plasmid HD1
K2BGH(rc)/shRNA10(rc)/sv40(rc)/CMV/Luc2_sv40/CMV/Kozag_hRluc_BGHBBa_K337035 pSMB_miTuner Plasmid HD2
K5BGH(rc)/shRNA10(rc)/sv40(rc)/RSV/Luc2_sv40/RSV/Kozag_hRluc_BGHBBa_K337040 pSMB_miTuner Plasmid HD5
K6BGH(rc)/shRNA10(rc)/sv40(rc)/CMV/RSV/Luc2_sv40/RSV/Kozag_hRluc_BGHBBa_K337042 pSMB_miTuner Plasmid HD6
K7BGH(rc)/shRNA10(rc)/RSV(rc)/CMV/Luc2_sv40/RSV/Kozag_hRluc_BGHBBa_K337044 pSMB_miTuner Plasmid HD7
K8BGH(rc)/shRNA10(rc)/RSV(rc)/CMV_TetO2/Luc2_sv40/RSV/Kozag_hRluc_BGHBBa_K337046 pSMB_miTuner Plasmid HD8