BioBrickBox

In 2012, the iGEM-team LMU-Munich began the task to develop essential Biobricks, like vectors, promoters, reporters and affinity tags, especially suited for the use in B. subtilis in order to establish a new chassis in the E. coli dominated world of iGEM. Even after the finale of the iGEM competition in 2012, the project was pursued further since its importance for synthetic biology and resulted in a publication in the Journal of Biological Engineering about this so called Bacillus BioBrick Box (B4)

This year, our team aims to enhance this Bacillus BioBrick Box by adding new parts, like different colored fluorescent proteins or a whole new cloning strategy.

Resistance Free Cloning

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Background

The upp gene from Bacillus subtilis W168 encodes for a Uracilphosphoribosyl transferase (UPRTase). Its key reaction in uracil salvage is the reaction of a uracil molecule with a 5'-phosphoribosyl-a-1- pyrophosphate (PRPP) molecule, resulting in the formation of UMP. A second locus, the pyrR Gene, encoding a second UPRTase has been identified. However it has been shown, that the UPRTase derived from pyrR locus has an influence on overall UPRTase activity < 1 %. (J Martinussen, P Glaser, P S Andersen and H H Saxild J. Bacteriol. 1995, 177(1):271. ) This makes the upp gene derived UPRTase the only physiologically relevant catalyst for UPRTase activity. Exposure to the pyrimidine analogue 5-Fluorouracil UPRTase results in production of 5-fluoro-dUMP, a very potent inhibitor of the thymidylate synthase (Neuhard, J. (1983) Utilization of preformed pyrimidine bases and nucleosides. In Metabolism of Nucleotides, Nucleosides and Nucleobases in Microorganisms. Munch- Petersen, A. (eds). New York: Academic Press, pp. 95– 148. ). As a result 5-FU is toxic to the Bacillus subtilis W168 strain. B. subtilis 5FU-resistant (5FUR) mutants selected on low drug concentration (10 mM 5FU) are UPRTase-defective. (Nygaard, P. (1993) Purine and pyrimidine salvage pathways. In Bacillus Subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics. Sonenshein, A.L., Hoch, J.A. and Losick, R., (eds). Washington, DC: American Society for Microbiology, pp. 359–378. ) This has made upp a go to choice for negative selection in combination with an B. subtilis W168 ?upp strain. So far it has been used to make clean in-frame deletions and point mutations. (A new mutation delivery system for genome-scale approaches in Bacillus subtilis Céline Fabret,† S. Dusko Ehrlich and Philippe Noirot* Génétique Microbienne, INRA, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France. )

However to our knowledge no application using upp for clean insertions has been established so far.

The I-SceI Restricition endonuclease has a highly specific recognition sequence of 18 nucleotides. No such sequence is present in the W168 strain. It creates a double strand break at targetet location, which leads to an increased rate of repair at the specific site. By this the rate of homologous recombination has already been indreased by an factor of 100x. (Establishment of a Markerless Mutation Delivery System in Bacillus subtilis Stimulated by a Double-Strand Break in the Chromosome Ting Shi1,2,3,4., Guanglu Wang1,2,3,4., Zhiwen Wang1,2,3,4*, Jing Fu1,2,3,4, Tao Chen1,2,3,4, Xueming Zhao1,2,3,4 )

Design

The Plan was to restructure the BioBrick compatible, integrative Bacillus subtilis vectors pBS1C, pBS2E and pBS4S in a fashion that leads to deletion of the antibiotic resistance after Insertion of a gene of interest has been made. Integration of those basic vectors is achieved via homologous recombination between the lacA/thrC/amyE locus and the corresponding up and down fragments on the vector. This leads to an insertion of the Gene of interest within the RCF25 compatible multiple cloning site and the resistance for positive selection (MLS/Spec/CAM).

We wanted to add the upp-cassette, containing an I-SceIsite, as well as an additional up/down fragment to the vector. The desired vectors are presented in Fig. 1.. The upp-cassette will allow negative selection in 5FU media. The contained I-SceI site will be cut by the I-SceI restriction endonuclease encoded on helper plasmid pEBS-cop1. (Figure)

Cloning Strategy

Results

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