bioRxiv preprint doi: https://doi.org/10.1101/392423; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 A Bacterial Expression Vector Archive (BEVA) for flexible modular 2 assembly of Golden Gate-compatible vectors 3 4 5 Barney A. Geddes*, Marcela A. Mendoza-Suárez*, Philip S. Poole# 6 7 Department of Plant Sciences, University of Oxford, Oxford, UK. 8 9 10 Running Head: Modular assembly of bacterial vectors 11 12 *These authors contributed equally to this work 13 # Address correspondence to: Philip Poole, [email protected] 14 15 1 bioRxiv preprint doi: https://doi.org/10.1101/392423; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 16 ABSTRACT We present a Bacterial Expression Vector Archive (BEVA) for the 17 modular assembly of bacterial vectors compatible with both traditional and 18 Golden Gate cloning, utilizing the Type IIS restriction enzyme Esp3I. Ideal for 19 synthetic biology and other applications, this modular system allows a rapid, low- 20 cost assembly of new vectors tailored to specific tasks. To demonstrate the 21 potential of the system three example vectors were constructed and tested. Golden 22 Gate level 1 vectors; pOGG024, with a broad-host range and high copy number 23 was used for gene expression in laboratory-cultured Rhizobium leguminosarum, 24 and pOGG026, with a broad-host range a lower copy number and excellent 25 stability, even in the absence of antibiotic selection. The application of pOGG026 26 is demonstrated in environmental samples by bacterial gene expression in 27 nitrogen-fixing nodules on pea plants roots formed by R. leguminosarum. Finally, 28 the level 2 cloning vector pOGG216 is a broad-host range, medium copy number, 29 for which we demonstrate an application by constructing a dual reporter plasmid 30 expressing green and red fluorescent proteins. 31 32 IMPORTANCE Modular assembly is powerful as it allows easy combining of 33 different components from a library of parts. In designing a modular vector 34 assembly system, the key constituent parts (and modules) are; an origin of 35 plasmid replication, antibiotic resistance marker(s), cloning site(s), together with 36 additional accessory modules as required. In an ideal vector, the size of each 37 module would be minimized, and this we have addressed. We have designed such 38 a vector assembly system by utilizing the Type IIS restriction enzyme Esp3I and 2 bioRxiv preprint doi: https://doi.org/10.1101/392423; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 39 have demonstrated its use for Golden Gate cloning in Escherichia coli. An 40 important attribute of this modular vector assembly is that using the principles 41 outlined here, new modules for specific applications, e.g. origin of replication for 42 plasmids in other bacteria, can easily be designed. It is hoped that this vector 43 construction system will be expanded by the scientific community over time by 44 creation of novel modules through an open source approach. 45 46 KEYWORDS Golden Gate, level 1, level 2, modular assembly, cloning vector, 47 shuttle vector, broad host-range plasmid, open source, plasmid design. 3 bioRxiv preprint doi: https://doi.org/10.1101/392423; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 48 Over the last decade, synthetic biology has emerged as a powerful tool for 49 biotechnology and basic science research (1). Coupled with rapidly improving 50 DNA synthesis technologies, one of the key advances that has potentiated the 51 rapid expansion of synthetic biology is new DNA assembly technologies (2). The 52 ability for synthetic biologists to rapidly assemble and test new designs at low 53 cost is invaluable to progress in a wide-range of life sciences research. 54 Applications for this range from investigating new antibody production with 55 higher success rates, to increased food production minimizing the carbon footprint 56 with the effective transfer of nitrogen fixation between bacterial species (3). 57 Golden Gate cloning is a DNA assembly technology that utilizes type IIS 58 restriction enzymes to assemble multiple fragments of DNA in a linear order (4). 59 Since type IIS restriction endonucleases cleave outside of their recognition site, 60 the nucleotides in the cut-site of the enzyme are not discriminated and can be 61 tailored to suit. This permits assembly of multiple fragments of DNA in a 62 directional, linear order by using unique cut/ligation sites between each fragment, 63 all of which can be cut by the same type IIS restriction enzyme. The two most 64 commonly used Golden Gate enzymes are BsaI and BpiI, each has a 6 base-pair 65 (bp) recognition site and a 4 bp sticky-end cut site (4) . The ability of these 66 enzymes to cleave outside of their recognition site and careful design of 67 compatible overhanging sequences allows repeated rounds of cutting followed by 68 ligation to proceed towards a stable assembly product which remains undigested 69 as it lacks enzyme recognition sites (due to the inward orientation of the type IIS 70 recognition sites at the far 5’ and 3’ ends of modules) (5). This approach has 4 bioRxiv preprint doi: https://doi.org/10.1101/392423; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 71 given a massive improvement in the efficiency of assembly compared to 72 traditional rounds of restriction/ligation cloning and has been used to assemble at 73 least nine fragments in linear order, with 90% of transformed bacterial colonies 74 containing correct products (5). 75 Golden Gate cloning platforms have been developed for plants, fungi and 76 the bacterium Escherichia coli (6–9). However, in a wide-range of bacteria, 77 vectors for cloning using Golden Gate tools are not available. The ability to use 78 the most up-to-date molecular tools is crucially important for agricultural 79 biotechnology. Here we describe the development of a system for modular 80 assembly of Golden Gate-compatible cloning broad-host range vectors from a 81 library of vector parts. As these vectors are able to replicate in E. coli, this 82 facilitates easy genetic manipulation since following plasmid construction, as the 83 plasmids constructed can be transferred easily to other bacterial species. We 84 demonstrate the efficacy of this system by constructing several vectors, analogous 85 to useful traditional cloning broad-host range vectors but that are now Golden 86 Gate-compatible: level 1 cloning broad-host range vectors pOGG024, a medium 87 copy plasmid for gene expression for in vitro grown bacteria, and pOGG026, a 88 low copy number stable vectors for stable environmental gene expression in 89 environmental samples where no antibiotic selection is applied. We also describe 90 the construction through this modular vector system of a level 2 broad-host range 91 vector pOGG216. 92 93 RESULTS AND DISCUSSION 5 bioRxiv preprint doi: https://doi.org/10.1101/392423; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 94 Modular vector assembly. A new standardized system for vector assembly was 95 designed based on Golden Gate cloning and the MoClo system described by 96 Weber et al. 2011 (4). We have designed a bacterial vector assembly system with 97 a series of discrete key modules. In order to do this we took advantage of the 98 modularization and miniaturization of bacterial vector modules in the Standard 99 European Vector Archive (SEVA) (10, 11). In SEVA these modules are arranged 100 in a predefined order and assembled using standard restriction enzymes and 101 standard restriction/ligation reactions. We have conserved this organization in the 102 modular vector assembly system: position 1 is the cloning site(s), position 2 is the 103 antibiotic resistance cassette, position 3 is origins of replication and transfer. 104 Three more positions (4 to 6) have been reserved for additional accessory 105 modules for specific applications. Endlinkers containing terminators (ELT) are 106 used to circularize the plasmid by connecting the final position used to position 1 107 (Fig. 1A). We incorporated Golden Gate level 1 and level 2 cloning sites into 108 position 1 to make them compatible with the MoClo Golden Gate cloning system 109 (Fig. 1B). Each vector construction module is flanked with inward-facing 110 recognition sites for the type IIS restriction enzyme Esp3I and the cut sites, giving 111 overhangs with sequences specific for each module (fusion sites shown in Table 112 1), allow directional linear assembly in a one-pot reaction such as used in Golden 113 Gate cloning (4). 114 115 Vector module design. We assigned modules a universal position for linear 116 assembly based on function; position 1 - cloning site(s), position 2 - antibiotic 6 bioRxiv preprint doi: https://doi.org/10.1101/392423; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.