# 🥈 Engineering # Spirulina homologous recombination It has been learned from the literature (Dehghani,J 2018; Jeamton,W 2017;Jester,B 2021) that Spirulina transformation is performed successfully only if the recombinant DNA is **inserted into the genome.** The plasmids can survive for a short time only in the presence of costly nuclease inhibitors (Dehghani, J 2018). The genome integration can be achieved through homologous or site-specific recombination. > Homologous recombination overview and requirements.
Homologous recombination is a type of genetic recombination. During this process exchange of DNA sequence between two similar or identical sequences occurs. Cells often use this type of recombination to repair one- or two-stand gaps. In genetic engineering it is used to insert sequences into genome of organisms. A succesful insertion can be achieved by flanking the inserting genetic elements with sequences of the recombination locus. > Site-specific recombination overview and requirements. Site-specific recombination is a mechanism of DNA mutagenesis, commonly used in Genetic Engineering, borrowed from bacteriophages. It allows to swap rather big fragments of genetic material, between two short genetic sequences. Enzymes that cause this type of mutations are highly specific. ### Insert assembly According to our goal of creating a Spirulina-based chassis organism, we have to create a system such as following: - the strain would be easily transformed, ideally allowing for a foolproof protocol - Auxiliary parts would require minimum space in order to maximize the insert size. - recombination machinery ("drivers") would have to be already installed in Spirulina genome We have come up with a way to meet all of the criteria above. To be easy to use, the final system should contain the site-specific bacterial recombination sites and a recombinase, and the insert will be required to have the flanking phage recombination sites. The recombinase cannot be delivered with the insert DNA, as it wouldn't have time to express and function, thus, it has to be there beforehand. To enrich the Spirulina with the recombinase, we have to perform a **prior round of homologous recombination**, inserting the site-specific recombination sites, selection marker and the Bxb1 recombinase. Homologous recombination has been shown to insert up to 7 kbp of linear DNA. Our work was hampered by the fact that there are no assembled and annotated genomes of the available strains, and we had to include many additional steps. In order to test the Spirulina competence we have assembled the insert, which comprises 9 parts. ![https://static.igem.org/mediawiki/2021/e/e7/T--LMSU--Team_LMSU_eng1%283%29.jpeg] Genes and promoters were PCR amplified from the available commercial plasmids (kindly provided by Evrogen and IPPRAS Laboratory of Microalgae Ecophysiology); smaller fragments were obtained by annealing oligos (supplied by Evrogen). ~|| ![https://static.igem.org/mediawiki/2021/7/7d/T--LMSU--ControlABR.tiff](Antibiotic resistance control, agarized Zarrouk medium, see /Protocols)| ![https://static.igem.org/mediawiki/2021/a/a6/T--LMSU--KanABR.tiff](Antibiotic resistance control, agarized Zarrouk medium with various concentrations of kanamycin, see /Protocols)|| A strain of Spirulina resistant to kanamycin was selected from the IPPAS collection. Initially we hoped to get the sequence of the gene using degenerate primers, then design specific primers to conduct a reverse PCR to attain the homologous arms by amplifying the DNA of regions upstream and downstream from the gene itself. Today we can present you the sequence and the inferred homology of the kanamycin resistance gene, which can be found in the /Results section. Though, this gene was observed to be in an operon with another uncharacterised ORF; therefore, we implemented the operon promoter in the end of our insert in order to not disturb expression and function of the unknown component. Due to uncertainties about the swiftness and overall availability of sponsor synthetic DNA or GoldenGate(Kucera, Cantor, NEB 2018) enzymes we decided to assemble the insert from scratch, using linear PCR amplified or annealed dsDNA; this decision turned out to be a mistake, on which we have elaborated. **We do not recommend anyone to try to assemble 3kb of dsDNA from linear fragments and relying on 9 consecutive restriction-ligation reactions due to analysis complications and significant DNA losses during purification.** Additionally, annealing attB sites yielded byproducts which interfered with the reaction and practically halted the assembly process. ![https://static.igem.org/mediawiki/2021/0/09/T--LMSU--eng3.jpeg](This gel shows pairs of annealed attB sites with (even lanes) or without (odd lanes) purification step. As it can be seen, the smear persists, which means that attB sites could not be obtained in that way, possibly because of their symmetry) Therefore, we have outlined several issues, which have to be addressed next year. 1. The excess of steps and the absence of final result showed the unviability of chosen assembly method. We will fuse the parts in advance using the gene synthesis options, or, despite being noticeably expensive, assemble the insert using Gibson or GoldenGate methods; 2. The Bxb1 recombinase (BBa_K1039009) shall replace the YFP gene after the transformation step is established; 3. The site of insertion was chosen without much alternative; however, upon getting the genome sequence, we might want to inspect the chosen region more closely, as well as search for other options. ### Plasmid and protein half-life expectancy The homologous recombination can be induced by introducing the dsDNA break or nick in the place of desired mutation, e.g. using CRISPR/Cas system or another endonuclease. To estimate the prior chances of implementing such a mechanism, we provide the experimental design scheme for us and any other team to apply and try to tinker and elaborate upon. Using the available genetic manipulation method, an attempt to transform *A. platensis* with a plasmid containing a reporter YFP gene and a selection marker ampicillin was performed. The plasmid pop-in mechanism is mainly speculative. After the removal of the selection driver (e.g. replating in a fresh medium batch), the plasmid might be popped out. As it does, we intend to estimate the half-life periods of plasmid DNA and synthesised heterologous protein, which will affect the effectiveness of later steps, that involve integrase-mediated recombination activity. ![https://static.igem.org/mediawiki/2021/7/76/T--LMSU--enga.png] ![https://static.igem.org/mediawiki/2021/2/20/T--LMSU--engb.png] The chosen transformation protocol relied on the competence induced by co-cultivation (Jester, Zhao, 2021), but after the addition of the antibiotic no cells survived. Future attempts will employ both positive and negative selection, allowing for plasmid pop-in/pop-out and studying the way for inducing competence and prolongation of plasmid life. ![https://static.igem.org/mediawiki/2021/5/5e/T--LMSU--Team_LMSU_eng2%283%29.jpeg] # Optogenetics ### Assembly of optogenetic construction Our optogenetic system consists of two main components: photosensitive protein BphP1 and its partnering protein QPAS1 which binds to specific promoter and blocks transcription via the attached Gal4 factor. Our final goal was assembling and analysis of constructions, consisting of all aforementioned elements. ![https://static.igem.org/mediawiki/2021/e/e9/T--LMSU--engc.png](**а.** Scheme of optogenetiс сonstruction **b.** Identical construction with YFP reporter gene) This system comprises 2 genetic parts: - 2-protein Regulatory system - Controlled gene under QPAS1-Gal4-dependent promoter. We also plan to assemble optogenetic part without the regulated gene, as the genes might have to be spaced out within the genome or the assembly process might include consecutive insertion of regulating and regulated parts. We consulted other phototroph teams (Madrid and Marburg) to get advice on assembling of such complex genetic constructions. Ultimately, we concluded that we would use Golden Gate protocol with PhytoBrick standards(Patron et al. 2015). The design, underlying our assembling protocol, was generously given us by Marburg 2018. ![https://static.igem.org/mediawiki/2021/9/94/T--LMSU--Team_LMSU_eng3%282%29.jpeg](Assembly standard with scar sites) We used some variations of pUC-kanR-LacZ plasmid as vectors for lvl1 constructions and some variations of pUC-kanR-LacZ for lvl2 constructions. All plasmids differed from each other as in some of them additional BsmBI sites had been inserted. Plasmids with a single BsmBI site were used for assembling of constructions, with were subsequently used for insertion into lvl2 constructions. To achieve that linker without BsmBI site was added to the reaction mix during assembling of lvl1 constructions. This modification replaced addition of of two linkers into reaction mix and it was required as long as standard of used plasmid did not concur with PhytoBrick standard. We gave a priority to the assembly standard and, therefore, had to add some additional steps in vector processing. It is important to mention, that such plasmid collection will simplify the assembling of two-component lvl2 systems so that no additional linkers will be required. ![https://static.igem.org/mediawiki/2021/thumb/d/d5/T--LMSU--eng9.png/800px-T--LMSU--eng9.png](Proposed implementations of plasmids) pUC-kanR-LacZ was chosen as a backbone for lvl2 constructions. It was also modified by inserting both of the BsmBI sites. ### Design of QPAS-Gal4-dependent promoters We picked up an eukaryotic regulator to make the system unique for bacteria. Therefore, onу of the main goals was to design a promoter which will function in *E. coli* efficiently and will be repressed by Gal4. We chose Anderson promoters (Parts form BBa_J23100 to BBa_J23119) as a basis. For the primary experiment pj23119 (BBa_J23119) promoter was chosen and modified with insertion of consensus sequence of Gal4 binding site from *Saccharomyces cerevisiae*. This sequence is located between GACA-box and TATA-box. As we had no prediction accorarding to the activity of the newly generated promoters, we decided to analize two types of sites: one containing only 3+3 crucial nucleotides which are directly recognized by HTH domains of Gal4 (p119-G4-n11) and the other with the whole 3+11+3 sequence of Gal4 site (p119-G4). So if accoarding to sagnificant rearrangements p119-G4 were unrecognizable for bacteria or had too small activity we could check less transformed option of p119-G4-n11 that could still de dependant from Gal4 and also be recognisable for *E. coli*. ![https://static.igem.org/mediawiki/2021/b/bb/T--LMSU--Team_LMSU_eng4%282%29.jpeg] Therefore, we had to test both base activity of these promoters and the inhibition efficiency of Gal4 with them to get a proper characterisation of these parts. The baseline activity was tested by YFP output with strong RBS used (BBa_J34801). We compared the relative activity of modified promoters with the original J23119 promoter. We tested the efficiency of Gal4 with chosen promoters in a given construction. ![https://static.igem.org/mediawiki/2021/2/2d/T--LMSU--eng12.png] As a negative control also original promoter was taken. Additionally, we constructed the same system but instead of J23119 dummy promoter was placed to confirm that there is presented a considerable difference between active QPAS1-Gal4 and its absence in the same conditionals. Thus we get two new promoters dependant on Gal4 that can be used in procaryotic organisms that we consider to be a significant part improvement. Our further step is to try other Anderson promoters to create a library of bacterial Gal4 dependant promoters that there could be more chance to select the best fitting element for different chassies. ### Improving efficiency and sustainability of the optogenetic system It was shown in original articles that in eukaryotic cells even under inhibitory signal of red light the non-zero level of fluorescence is observed. However, the baseline activity of QPAS1-Gal4 can be decreased by fusing it to BphP1 — the additional light-sensitive protein that can increase the system specificity. We decided to look for a protein that reacts to a different wavelentgh and can work during the growth period of *Arthrospira,* which led us to blue-light sensitive proteins. From the variety of optogenetic systems of blue-light proteins, we chose LOV-domain containing protein BcLOV4 from *Botrytis cinerea* as it has a unique trait to be recruited to a membrane under blue light. It creates a specific light switchable "compartmentalisation" for proteins. Hereby, by fusing BphP1 with BcLOV4 we add the second tool to control transcription by blue light(Glantz, 2018). ![https://static.igem.org/mediawiki/2021/a/a7/T--LMSU--eng13.png] Finally, we designed two extra constructions with a whole optogenetic system but with BcLOV4 attached to BphP1. ![https://static.igem.org/mediawiki/2021/6/6b/T--LMSU--eng7.png] ### Light-sensitive complex localisation analysis Going further from modelling of BcLOV4-BphP1 we planned to study the spatial distribution of this chimeric protein in cells under different light conditions using fluorescent microscopy. If we excite cells with 450 nm light, the BcLOV4 will bind to the membrane, and FMN incorporated in BcLOV4 will fluoresce with the highest emission intensity around 500 nm. ![https://static.igem.org/mediawiki/2021/2/25/T--LMSU--eng17.png]