Accurate DNA assembly and genome engineering with optimized uracil excision cloning

Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of <i>gfp</i> and a four-gene pathway that produces β-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly facilitating the advanced engineering of robust cell factories

(+)-Geodin from Aspergillus terreus.

The fungal metabolite (+)-geodin [systematic name: (2R)-methyl 5,7-dichloro-4-hydroxy-6'-methoxy-6-methyl-3,4'-dioxospiro[benzofuran-2,1'-cyclohexa-2',5'-diene]-2'-carboxylate], C(17)H(12)Cl(2)O(7), was isolated from Aspergillus terreus. The crystal structure contains two independent molecules in the asymmetric unit. Molecules denoted 1 interact through O-H...O hydrogen bonds creating chains of molecules parallel to the crystallographic 2(1) screw axis. Molecules denoted 2 interact through an O...Cl halogen bond, also creating chains of molecules parallel to the crystallographic 2(1) screw axis. Molecules 1 and 2 interact through another O...Cl halogen bond. The two molecules are similar but molecules 2 have a slightly more planar cyclohexadiene ring than molecules 1. The absolute structure of (+)-geodin has been unequivocally assigned with the spiro centre having the R configuration in both molecules. The structurally related (+)-griseofulvin has an S configuration at the spiro centre, a difference of potential biological and biosynthetic relevance

A proof-of-concept study for the design of a VLP-based combinatorial HPV and placental malaria vaccine

Abstract In Africa, cervical cancer and placental malaria (PM) are a major public health concern. There is currently no available PM vaccine and the marketed Human Papillomavirus (HPV) vaccines are prohibitively expensive. The idea of a combinatorial HPV and PM vaccine is attractive because the target population for vaccination against both diseases, adolescent girls, would be overlapping in Sub-Saharan Africa. Here we demonstrate proof-of-concept for a combinatorial vaccine utilizing the AP205 capsid-based virus-like particle (VLP) designed to simultaneously display two clinically relevant antigens (the HPV RG1 epitope and the VAR2CSA PM antigen). Three distinct combinatorial VLPs were produced displaying one, two or five concatenated RG1 epitopes without obstructing the VLP’s capacity to form. Co-display of VAR2CSA was achieved through a split-protein Tag/Catcher interaction without hampering the vaccine stability. Vaccination with the combinatorial vaccine(s) was able to reduce HPV infection in vivo and induce anti-VAR2CSA IgG antibodies, which inhibited binding between native VAR2CSA expressed on infected red blood cells and chondroitin sulfate A in an in vitro binding-inhibition assay. These results show that the Tag/Catcher AP205 VLP system can be exploited to make a combinatorial vaccine capable of eliciting antibodies with dual specificity

A VAR2CSA:CSP conjugate capable of inducing dual specificity antibody responses

Background: Vaccine antigens targeting specific P. falciparum parasite stages are under pre-clinical and clinical development. It seems plausible that vaccine with multiple specificities will offer higher protection. With this hypothesis, we exploited the Spy- Tag/SpyCatcher conjugation system to make a, post expression, dual antigen conjugate vaccine, comprising two clinically tested antigen candidates (CSP and VAR2CSA).Methods: The DBL1x-DBL2x-ID2a region of VAR2CSA was genetically fused with SpyTag at N-terminus. The full-length CSP antigen was genetically fused to C-terminal SpyCatcher peptide. The covalent interaction between SpyTag/ SpyCatcher enables the formation of DBL1x-DBL2x-ID2a:CSP conjugate vaccine. Immunogenicity and quality of antibody responses induced by the conjugate vaccine, as well as a control CSP-SpyCatcher vaccine, was tested in BALB/c mice.Results: Serum samples obtained from mice immunized with the conjugate vaccine were able to recognize both untagged DBL1x-DBL2x-ID2a as well as CSP antigen. Moreover, the geometric mean anti-CSP antibody titer was 1.9-fold higher in serum (at day 35 and 55 post-first immunization) from mice immunized with the conjugate vaccine, as compared to mice receiving the control vaccine.Conclusion: The data obtained in this study serves as proof-of-concept for the simultaneous induction of antibodies directed against individual antigen components in a dual stage anti-malaria vaccine.Keywords: Malaria vaccine, Circumsporozoite protein, VAR2CSA, CSP SpyCatcher, SpyTag-DBL1x-DBL2x-ID2a, bacterial superglue, DBL1x-DBL2x-ID2a:CSP conjugat