V Fig.2: A Individual genes

Transcription

1 The ACEMBL - flexbac-ec Kit - A VERSATILE AND CONVENIENT DONOR - ACCEPTOR BASED ASSEMBLY CLONING AND RECOMBINEERING SYSTEM FOR MULTI-GENE EXPRESSION IN E. COLI Multiplex expression of proteins are one of the challenges of synthetic molecular technologies since years. In functional formations of proteins these are interrelated by specific binding of the individual proteins to each other forming functional complexes or proteins are functionally related by series of biochemical reactions forming pathways interrelated e.g. by logically ordered series and networks of bio-catalysts able for creating natural and in case of biotransformative activities artificial compounds of one of the enzyme catalysts involved. Fig.1: Multiple Expression Cassette Concept. The assembly technology (flexbac-e. coli) is based on expression optimized and tuned cassettes of each the individual expression units. Calculating your optimal expression is supported by ATG s evomag the multi-objective - in silico evolution software package for Donor Acceptor based DNA-assembly systems (see APPENDIX). The flexbac-ec technoloy can be used for both (1) Serial integration of multiple genes and gene clusters forming poly-cistrons or clusters of several individually controlled genes organized as independent expression cassettes. (2) Parallel expression of gene variant libraries covering different functional features and compositions of the genes gene variants at individual positions. Gene variants can either designed for testing of different expression levels as a function of silent mutation gene modulations aiming on the improvement of formal-operational expression features or for functional relevant gene product features (see APPENDIX) Fig.2: A Individual genes as well as expression cassette units can be assembled via the SLIC (sequence and ligase independent cloning technology). The principle uses the T4- DNA polymerase 3 ->5 exonuclease activity for generating longer 5 - protruding single stranded DNA termini with adhesion sufficiently strong for keeping the joint strands together.

2 Fig2: B Expression units can be assembled via homing nuclease, HN cycling (see also APPEN- DIX) using iteratively regenerating BstXI-Restriction-sites. Expression boxes are liberated from DONOR-vectors by homing nuclease restriction and a special BstXI site. The resulting DNAs can be assembled via an iterative process cycling process where the BstXI site is subsequently regenerated in the corrrect orientation but the HN-site not. DONORs of higher order can be generated

3 Fig.3. Assembly of Donor Acceptor Constructs. Via the Cre/ Lox sites of Donor and Acceptor Plasmids there is a pathway for the fusion of independent gene cluster constructions into E. coli cells for stable maintainance there. For combinatorial molecular biology this ia another possibility for creating diversity by recombination of different constructs in case these were initially designed as different variant libraries in the experimental setting for achieving gene cluster variants for selection. Advantages OF flexbac-ec ASSEMBLY CLONING flexbac-ec is a 3rd generation multi-gene expression system for complex production in E. coli, created at the European Molecular Biology Laboratory EMBL, at Grenoble. flexbac-ec can be applied both manually and also in an automated setup by using a liquid handling workstation. flexbac-ec applies tandem recombination steps for rapidly assembling many genes into multigene expression cassettes. These can be single or poly-cistronic expression modules, or a combination of these elements. flexbac-ec also offers the option to employ conventional approaches involving restriction enzymes and ligases if desired, which may be the methods of choice in laboratories not familiar with recombination approaches. The following strategies for multigene - assembly and expression are provided by the flex- BAC-EC system: (1) Single gene insertions into vectors (recombination or restriction/ligation) (2) Multigene assembly into a poly-cistron (recombination or restriction/ligation) (3) Multigene assembly using BstX/ homing endonuclease cyclings (4) Multigene plasmid fusion by Cre-LoxP reaction (5) Multigene expression by co-transformation of compatible ori (6) SLIC Sub-cloning using T4 DNA polymerase treated inserts with RecA Sequence and ligation independent cloning sites assembly LIC-sites calculated by evomag at ATG:biosynthetics These different strategies for assembling Genes and GeneClusters to assemblies of higher order can be used individually or in conjunction with each other and also different other methods, depending on the project and user. All cloning strategies can be accompanied by the ATG: evomag software package for calculating the optimal assembly strategies and construction features. In addition evomag supports your gene design in empirical expression and production optimization we can calculate you any gene variant of formal-operational good function. Set of five flexbac-ec vetors...,,, pace1 (ColE1 dependent plasmid & strain) bp 2. pace2 (ColE1 dependent plasmid & strain) bp 3. pdc (R6K - pir+ - dependent plasmid & strain) bp 4. pds (R6K - pir+ - dependent plasmid & strain) bp 5. pdk (R6K - pir+ - dependent plasmid & strain) bp - 3 -

4 The flexbac-ec for E. coli Kit Contains Plasmids & E. coli Strains ACEMBL: flexbac-ec: ACCEPTOR PLASMIDS (1) Sequence name - pace2 Acceptor - Ampicilline (2) Size (bp) 2652 (3) Vector type of delivery psv*: ColE1 derivative (standard) (5) Codon usage adaption none (6) Store in solution at least at 4 C Buffered at ph7,2 (7) Expiry date of storage 6 Month * psv: standard vector (1) Sequence name - pace2 Acceptor - Tetracycline (2) Size (bp) 2982 (3) Vector type of delivery psv*: ColE1 derivative (standard) (5) Codon usage adaption none (6) Store in solution at least at 4 C Buffered at ph7,2 (7) Expiry date of storage 6 Month * psv: standard vector ACEMBL - Pir + HC - E. coli cells... For high copy number propagation of Donor pdk, pds and pdc-cells. ACEMBL - Pir + LC - E. coli cells.. For low copy number propagation of Donor pdk, pds and pdc-cells

5 ACEMBL: flexbac-ec: DONOR PLASMIDS (1) Sequence name - pds Donor Plasmid - Spectinomycine resistant (2) Size (bp) 2077 (3) Vector type of delivery pnsv*r6kg - pir+ - dependent (5) Codon usage adaption (6) Store in solution at least at Buffered at ph7,2 (7) Expiry date of storage * pnsv: non standard vector (1) Sequence name - pdc Donor Plasmid - Chloramphenicol resistant (2) Size (bp) 2067 (3) Vector type of delivery pnsv*r6k - pir+ - dependent (5) Codon usage adaption (6) Store in solution at least at Buffered at ph7,2 (7) Expiry date of storage * pnsv: non standard vector (1) Sequence name - pdk Donor Plasmid - Kanamycine resistant (2) Size (bp) 2027 (3) Vector type of delivery pnsv*r6kg - pir+ - dependent (5) Codon usage adaption none (6) Store in solution at least at Buffered at ph7,2 (7) Expiry date of storage * pnsv: non standard vector - 5 -

6 Fig.3. flexbac-ec Sequentially applying the integration of genetic elements for setting up biocatalytical pathways like artificial biochemical and biotransformational pathways. Multiple integration elements containing genetic control elements GCE and coding sequences CDS of genes for systematically building up artificial biocatalytical pathway systems for natural biochemical and biotransformation processes. Ordering individual catalyst genes or catalyst variant libraries in a functionally logical fashion offers the opportunity for step by step analysing the catalytical parameters of each of the pairs of educt and product compounds for setting up highly efficient catalytical systems and optimizing it s properties. Fig.4. flexbac-ec Sequential gene integration for functional expression of multi-protein complexes for e.g. drug discovery and high throughput toxicity assays. In contrast to individual genes gene variant libraries can be expressed in parallel and combined to each other in order to find the most efficient combination of variants. For identification and integration of minimal artificial genetic elements (mage-elements) from MACs to MACS (Minimal Artificial Catalytical Systems) please consult

7 Fig.5. flexbac-ec Sequentially applying the Integration of Genetic Elements for multiple cloning of protein complexes. Multiple integration elements (MIE) containing genetic control elements and coding sequences (CDS) of desired genes forming protein complexes of desired function. The flexbac-ec multi-protein expression is best suitable for systematically analysing these protein complexes in the functional context of changes of parameters. Fig.6. flexbac-ec One of the mayor features of the flexbac-ec system is it s capability for automation. Multiple integration elements (MIE) containing standardized parts and coding sequences (CDS) of desired genes can be assembled to desired functional features of devices and systems. The flexbac-ec is best suitable for systematically creating combinatorial changes in functional contexts of sequential gene clusters built of multiple integration elements and in parallel processing functional libraries per position of individial multiple integration elements. flexbac-ec The MAGIC (Minimal Artificial Genomes Integration by Cycling) and Integrated Circuits concept of ATG:biosynthetics is the most important and powerful technology for applying flex- BAC-EC to it s highest sophistication

8 (1) Sequence name- pace1 Acceptor Plasmid - Ampicilline resistant (2) Size (bp) 2652 (3) Vector type of delivery psv*: ColE1 derivative (standard) (5) Codon usage adaption (6) Store in solution at least at Buffered at ph7,2 (7) Expiry date of storage * psv: standard vector Fig.7. evomag Software for optimization of formal-operational gene function parameters covers several molecular levels of gene expression in the cells. The different levels of in silico gene optimization are based on the newest scientific publications and are advanced in it s genetic algorithms creating gene populations of sequence variants which are optimized for creating the weighted best compromize of conflicting sequence parameters. Gene variants can be systematically tested in pairs of expression cassettes by cloning sequentially two variant libraries in line, in order to identify the best functional results. In combination with the mage (minimal artificial genetic elements data-base MAGIC) and MAGIC assembly of Genomes (Minimal Artificial Genomes - Integration Cloning and Integrated Circuits concept of ATG:biosynthetics calculating of highly functional artificial pathway systems is feasible). In this integrations for applying flexbac-ec we bring the most important and powerful moleculartechnologies to it s highest sophistication with our clients

9 APPENDIX CLICK to SLIC - Sequence & Ligation Independent Cloning Fig.A1. Integration CUT OFF - Sites are short stretches of DNA flanked by two unique restriction sites which can be cut out for to liberate the SLIC sites ready for to create single stranded hybridizationtermini. Without ligation reaction these can be annealed to assemble expression cassettes. LIC is a cloning method that makes use of annealing of single-stranded complementary overhangs on the target vector and a PCR-generated insert of at least 12 bases. Single-stranded overhangs can be generated by using T4 DNA polymerase and only one dntp in the reaction mix, leading to an equilibrium of 3'->5'-exonuclease and 5'->3'-polymerase activity at the site of the first occurrence of this nucleotide. By optimizing the reaction conditions, Li & Elledge (Nature Methods, 2007, 4 (3), ) could show that no special requirements are needed for this overlap region (e.g. absence of one of the nucleotides in the terminal region). The incubation is done with T4 DNA pol. for 30 min. and stopped by adding dctp to the reaction mix. After annealing of vector and insert, the mixture is used to transform E. coli. FigA2: LIC (Ligation Independent Cloning) sites are created by T4-DNA Polymerase 3 -exonuclease activity and stretches of DNA were one of the four nucleotides is missing. Out of coding sequences those sequence features are easy to realize whereas LIC-sites located in coding gene sequences need to be carefully calculated in order not to reduce the codon adaption index to an inacceptable level. ATG:biosynthetics evomag Software is suitable for calculating LIC sites in coding sequences for supporting your molecular strategies and design.slic (Sequence and Ligation Independent Cloning) In cloning systems which use fixed stretches of overlapping DNA outside of coding regions in dual vector systems like flexbac assembly of expression cassettes is feasible. ATG supports your molecular strategies and designs with enthusiasm. Designing synthetic Genes for SLIC cloning you can easily introduce the appropriate SLIC sites terminal flanking to the gene to be cloned. ACEMBL vectors contain already prepared SLIC sites. If you like to assemble coding sequences of genes with appropriate SLIC sites not pre-determined by the cloning system - we can calculate the optimal sites for you - with evomag. Simultaneously we can find the best compromize for optimizing expression of the gene to be assembled

10 Homing nuclease cycling BstXI - PI-SceI FigA3: BstXI Homing nuclease cycling (here PI-SceI) for the assembling of expression boxes. DONOR plasmid based homing nuclease sites serves for large uniqueness of the restriction fragment excission process in that only with a very limited probability interference with redundant sites occurs. The restriction overlap of the homing nuclease and the BstX site is designed for ligation compatibility. After the ligation reaction the homing nuclease site is destroyed, the BstX site is restored. This allowes cyclic cloning of expression cassettes and poly-cistrones. ATG supports your BIBLIOGRAPHY 1. Bieniossek C. et al (2009) Automated unrestricted multigene recombineering for multi-protein complex production. Nat Methods 6: Salis et al. (2009) Automated design of synthetic ribosome binding sites to control protein expression,nature Biotechnology, 27: Brandt et al. (2009) The native 3D organization of bacterial polysomes. Cell 136: Li M.Z. & Elledge S.J. (2007), Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC, Nature Methods 4: Doyle S.A. (2005), High-throughput cloning for proteomics research, Methods Mol. Biol 310: