# Team:IISER-Pune-India/FutureWork

<!DOCTYPE html> Future Work

# E. Coli

### Bidirectional Promoter Characterization

We plan to characterize the native bidirectional promoter of the csc operon which we submitted as part BBa_K3971006. We also created a composite part to characterize the activity of this promoter BBa_K3971024. Since the cscK and cscB genes are transcribed in the direction opposite to cscA, a bidirectional promoter jointly regulates both sets of genes in the operon[1].

We will characterize the activity of the promoter using fluorescent reporters (a yellow and blue fluorescent protein) which flank the promoter and their relative fluorescence will inform us about the strength of transcription in both directions. The characterization cassette for the promoter has been submitted as the composite part BBa_K3971024. Characterization of this promoter will allow us to compare its strength and activity with other known promoters, in order to help us choose an ideal promoter for optimal sucrose consumption and consequent butanol yields.

### Genetic engineering based on modeling results

Our dry lab team used OptKnock and Flux Scanning based on Enforced Objective Flux (FSEOF) in order to identify potential candidate genes and reactions to target in order to increase sucrose and butanol yields. In the future, we aim to perform these modifications in the lab and hope to optimize the co-culture and final yields to make the process industrially viable.

### Increasing butanol tolerance

After talking to Dr. Anand Ghosalkar from Praj industries, our team learned that enhancing butanol tolerance in E. coli would cause a significant impact on the current market and potentially surpass the current methods of sustainable production. We, therefore, looked for possible over expressions and deletions that could cause increased butanol tolerance. We came across certain genes that were capable of this[2]. We also came across a butanol efflux pump[3] in E. coli that was shown to increase the host’s tolerance to butanol. Read more about this on our proposed implementation page.

# S. elongatus

## Characterizing native promoters

### Construct assembly

We have shortlisted the native cyanobacterial promoters - Pcpc560[4], PcpcB-m6[5], PpsbA2[6], and PpsbA3[5],[6] to characterize in our novel chassis S. elongatus UTEX 2973 against some other commonly used bacterial promoters - J23119 (from the iGEM kit) and lacUV5 (present in the sucrose-exporting strains of S. elongatus UTEX 2973 that we received)[7].

We intend to incorporate a promoter into a cassette upstream of the fluorescent reporter sYFP2 and a terminator. This cassette is itself flanked by two sequences NS1A and NS1B (BBa_K3971009) which are homologous to the first genomic neutral site (NS1) of Synechococcus elongatus UTEX 2973, intended for homology-directed repair. We will prepare this composite part via extension and overlap PCR using the primers we have designed. This part will be integrated into the [8]pSL26808 plasmid. Note that the promoter is flanked by restriction sites, allowing us to excise and replace it with other promoters without repeating the complicated assembly step.

On the other side of the plasmid is the gene cpf1 coding for the Cas12a enzyme. Downstream on the gene are various spacers, and a lacZ cassette, flanked by Aar1 restriction sites. We will synthesize a short stretch of DNA (via overlap PCR of two oligos) to bind to a protospacer in NS1 of the genome. This sequence will be introduced into the plasmid in place of the lacZ cassette and will allow for a CRISPR-Cas12a-mediated targeted break of the genomic DNA. The homology arms NS1A and B will then allow for homology-directed repair of the genome to include our composite part.

A very similar principle applies to the overexpression of the native sps gene (previous work has expressed sps and spp genes from Synechocystis PCC 6803 in Synechococcus elongatus UTEX 2973). This gene will be incorporated into the pSL26808[8] plasmid, with homology arms for neutral site 3 of the S. elongatus genome, taken from the pSL30057[7].

### Triparental Mating

The UTEX 2973 strain is unfortunately not naturally transformable[9], posing a challenge to genetic engineering work. To characterize the promoters and sps and spp, we will need to introduce the plasmids via conjugation from E. coli DH5$$\alpha$$. The plasmid of interest - pSL2680 - will be transformed into this strain, which will then undergo Kanamycin screening and blue-white screening to select for transformants with lacZ replaced.

For biosecurity reasons, DH5$$\alpha$$ is typically not capable of forming a conjugation pilus. It thus needs to acquire an F-plasmid from a helper strain (for this purpose, we will use E. coli HB101). This will carry not only the F-plasmid but also other helper and mobilizer plasmids (pRL443 and pRL623)[10] needed for safe conjugative transfer of pSL2680 (our plasmids of interest) into UTEX 2973[11].

### Fluorescence-based characterization

We will grow S. elongatus at different light intensities (100-1500 $$\mu$$mol photons/m2/s) and different carbon dioxide concentrations (0.04%-5%). Under each such pair of conditions, we will characterize the promoter activity by measuring the fluorescence of sYFP2. We will also simulate a day-night cycle, in accordance with S. elongatus’s natural circadian rhythm, to see if it impacts the sustainability and stability of (i) the axenic culture and its sucrose production and (ii) the co-culture and its butanol production. This is of particular interest in the case of PpsbA24[4] and PpsbA34[4], which are promoters associated with the circadian rhythm. We will select a promoter yielding optimum performance and use it to express cscB in UTEX 2973.

## Characterizing O2 evolution by cyanobacteria using redox indicators

Since anaerobic conditions are vital for maximizing butanol yields, we also aim to monitor oxygen levels in the culture to ensure that the cultures are as close to anaerobic as possible, given the oxygen evolution by cyanobacteria which might make the culture microaerobic. To do this we plan to either use The Oxoid Anaerobic Indicator by Thermo Fisher Scientific[12]. It consists of a redox indicator solution in a laminated foil envelope that turns white from red under anaerobic conditions. We initially considered other redox indicators such as resazurin, however, based on inputs from our mentors we chose the Oxoid indicator, given that resazurin was unlikely to work under microaerobic conditions and required highly anaerobic conditions.

The results of this experiment will also inform us of the need to use oxygen scavengers such as ascorbic acid in order to maintain near anaerobic conditions in the culture[13].

# References

1. A transferable sucrose utilization approach for non-sucrose-utilizing Escherichia coli strains. Bruschi M, Boyes SJ, Sugiarto H, Nielsen LK, Vickers CE. Biotechnol Adv. 2012 Sep-Oct;30(5):1001-10. doi: 10.1016/j.biotechadv.2011.08.019. Epub 2011 Sep 1. 10.1016/j.biotechadv.2011.08.019 PubMed 21907272
2. Reyes, L. H., Almario, M. P., & Kao, K. C. (2011). Genomic library screens for genes involved in n-butanol tolerance in Escherichia coli. PloS one, 6(3), e17678.
3. Sergey Boyarskiy, Stephanie Davis López, Niwen Kong, Danielle Tullman-Ercek,Transcriptional feedback regulation of efflux protein expression for increased tolerance to and production of n-butanol,Metabolic Engineering,Volume 33,2016,Pages 130-137,ISSN 1096-7176
4. Zhou, J., Zhang, H., Meng, H., Zhu, Y., Bao, G., Zhang, Y., ... & Ma, Y. (2014). Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria. Scientific reports, 4(1), 1-6.
5. Sengupta, S., Jaiswal, D., Sengupta, A., Shah, S., Gadagkar, S., & Wangikar, P. P. (2020). Metabolic engineering of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for photoautotrophic production of succinic acid. Biotechnology for biofuels, 13, 1-18.
6. Li, S., Sun, T., Xu, C., Chen, L., & Zhang, W. (2018). Development and optimization of genetic toolboxes for a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973. Metabolic engineering, 48, 163-174.
7. Lin, P. C., Zhang, F., & Pakrasi, H. B. (2020). Enhanced production of sucrose in the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973. Scientific reports, 10(1), 1-8.
8. Ungerer, J., & Pakrasi, H. B. (2016). Cpf1 is a versatile tool for CRISPR genome editing across diverse species of cyanobacteria. Scientific reports, 6(1), 1-9.
9. Yu, J., Liberton, M., Cliften, P. et al. Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO2. Sci Rep 5, 8132 (2015). https://doi.org/10.1038/srep08132
10. Elhai, J., Vepritskiy, A., Muro-Pastor, A. M., Flores, E., & Wolk, C. P. (1997). Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120. Journal of bacteriology, 179(6), 1998-2005.
11. https://www.jove.com/t/60451/genetic-modification-cyanobacteria-conjugation-using-cyanogate
12. http://www.oxoid.com/uk/blue/prod_detail/prod_detail
13. Wagner, A. O., Markt, R., Mutschlechner, M., Lackner, N., Prem, E. M., Praeg, N., & Illmer, P. (2019). Medium preparation for the cultivation of microorganisms under strictly anaerobic/anoxic conditions. Journal of visualized experiments: JoVE, (150).