To genetically engineer cyanobacteria, we chose Synechococcus elongatus PCC 7942 as our engineering host. Our main strategy is to embark on gene double-crossover homologous recombination in S. elongatus PCC 7942 genome, which is the first cyanobacterial strain to be transformed by exogenous DNAs and is reliably transformable through natural uptake of extracellular DNAs.

　　First, we constructed a vector which is able to finish double-crossover homologous gene recombination in S. elongatus PCC 7942. The vector (pPIGBACK) contains 5’- and 3’-ends of the neutral site II (NSII) and an ampicillin resistance gene (AmpR) for antibiotic selection. Then we fused AmpR with double terminator, BBa_B0015, which is proved to be functional in cyanobacteria. Additionally, in order to easily manipulate DNAs for gene cloning and plasmid preparation in E. coli DH5α, the replication origin (ORI) of pBR322 was also introduced to make the plasmid vector replicable in E. coli. Then, in order to overexpress foreign genes in the cyanobacteria, the intrinsic promoter of Rubisco large subunit (PrbcL) was chosen as the target for vector construction. PrbcL regulates the expression of the most abundant proteins in photosynthetic species and has been proven to have a high activity to express foreign genes, so we chose PrbcL as the promoter of our pigment gene.¹

　　The strategy we chose to construct the vector is to fuse B0015 and AmpR together first. Secondly, we fused 5’- and 3’-ends of the neutral site II (NSII) with PBR322 replication origin (ORI) together. At last, we ligated two parts together. The vector (pPIGBACK) is used to transform into PCC7942 with the inserted pigment gene in our experiments. After mass reproduction in E. coli DH5α, PCC7942 were transformed through the uptake of plasmid DNAs extracted from E. coli DH5α. The transformed strains (transformants) were usually successfully obtained after 2 to 3 weeks and survived the ampicillin treatment.

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　　Indigoidine is a bacterial natural product with antioxidant and antimicrobial activities. Its bright blue color resembles the industrial dye indigo, thus representing a new natural blue dye that may find uses in industry. In nowadays studies, an Indigoidine synthetase Sc-IndC and an associated helper protein Sc-IndB were identified from Streptomyces chromofuscus ATCC 49982 and successfully expressed in Escherichia coli BAP1 to produce the blue pigment². The IndB gene codes for a putative phosphatase and the IndC gene codes for Indigoidine synthase. Together, these enzymes convert L-glutamine into Indigoidine. Recently, it has been shown that IndC alone can produce Indogoidine, and the inclusion of IndB expression in the system will increase yields significantly³.

　　As we know, L-Glutamine is the direct biosynthetic precursor of Indigoidine, and it is a key amino acid in primary metabolism and thus naturally exists in S. elongatus PCC7942. Because glutamine related products are already existed in S. elongatus PCC7942, we only need to activate the expression of Sc-IndC in S. elongatus PCC7942 which leads to the production of Indigoidine. However, due to the access difficulties of Streptomyces chromofuscus ATCC 49982, we decided to use the previous part for IndC, which has been submitted to the iGEM Parts Registry (BBa_K1152008)⁴. According to the part design, our Indigoidine gene comes from Photorhabdus luminescens laumondii TT01 (DSM15139).

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　　Zeaxanthin belongs to carotenoid family and is widely found in the nature. It is also a natural color making corns, carrots and marigolds yellow. Moreover, zeaxanthin is an essential nutrient substance to human’s eyes, and some healthy supplements are made of it. Most of green plants produce zeaxanthin as an intermediate in carotenoid pathway. However, some photosynthetic bacteria such as cyanobacteria lack of zeaxanthin. Therefore, we try to transform zeaxanthin-related genes to cyanobacteria to make them yellow⁶.

　　After paper study, we find nobody has done it before. Under our instructor’s help, we develop a way to make cyanobacteria yellow. We compare the complete carotenoid pathway with Synechococcus elongatus PCC 7942 whole genomic DNA on KEGG and we find every zeaxanthin-related gene is included in PCC 7942 genomic DNA except beta-carotene hydroxylase (crtZ) . And we find crtZ coding sequence with ribosome-binding site is an igem released part (BBa_I742158), which is from a plant pathogen, Pantoea ananatis⁵.

　　We had successfully transformed CrtZ to Escherichia coli to reproduce massively, and then transformed CrtZ with pPIGBACK to Synechococcus elongatus PCC 7942. After a week, the transformed Synechococcus elongatus PCC 7942 expressed more yellow than the control group. To test whether the photosynthetic efficiency is better, we used iodine to measure starch concentration and compare it with wild type.^{7, 8}

This figure is pPIGBACK-CrtZ transformants electrophoresis result. C1~C20 represents the pPIGBACK-CrtZ transformant clone 1 to clone 20, and M represnets 1 kb marker.

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　　Melanin, a biological pigment widely found in terrestrial flora and fauna, is a complex oxidation product of amino acid tyrosine. Melanin plays diverse roles in a myriad of organisms. As far as the human is concerned, melanin is the primary determinant of human skin color and pupils or irises of the eyes. It is also an important signal molecule in the human neural system. For microorganisms, melanin would protect them by against ultraviolet radiation effect from sunlight, which is detrimental to most of the organisms.

　　Due to the fact that the dark pigment derived from MelA gene has extensive wavelength absorbance, we decided to transform MelA gene combined with particular constitute promoter into Synechococcus elongates PCC 7942 to measure the growth curve and photosynthetic efficiency of it. Based on the well-elaborated procedure provide by IGEM Tokyo Tech 2009⁹, we had intended to replicate their experiment to produce melanin massively first in E. coli, but failed to succeed due to inconsistence of DNA sequence. Therefore, we had no alternative but to turn to DNA synthesis and directly transform MelA gene ligated with our backbone into microalgae.

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　　Astaxanthin is a high value and natural pink pigment which can be found in microalgae, yeast and some sea creatures. It’s special due to its antioxidant activity and has been suggested to be beneficial in cardiovascular, immune, inflammatory and neurodegenerative diseases and skin health. Although it has lots of benefits, astaxanthin is still a product result in a minority amount in the carotenoid synthesis pathway compare with other carotenoid families and yet, the artificial chemical synthesis cost high and result in the least production.

　　Astaxanthin synthesis does not naturally exist in the S. elongatus PCC7942. But fortunately, after paper research, we found out that S. elongatus PCC7942 has a similar pathway with other microalgae which can synthesize astaxanthin, and the only different is, S. elongatus PCC7942 lack of two necessary gene: beta-carotene ketolase (crtW) and beta-carotene hydroxylase (crtZ) to undergo this pathway.^{10, 11} Thus, we use IDT to synthesis these two genes and construct it on pPIGBACK, a vector which can express the carrying genes in S. elongatus PCC7942.

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　　Lycopene is a bright red carotene and carotenoid pigment found in tomatoes and other red fruits and vegetables. In photosynthesis, Lycopene plays a role in absorbing sunlight (from the wavelength about 460nm to 500nm)¹² and transferring the energies to Chlorophyll through electrons. It can also protect Chlorophyll from the damage of sunlight. The structure of Lycopene makes it a potent antioxidant among various common carotenoids. In Ames test, referring to testing whether a given chemical would cause mutations in the DNA, Lycopene shows its great ability to trap singlet oxygen and reduce mutagenesis.¹³ Numerous studies have also shown that Lycopene has a significant contribution to prevent cardiovascular disease, diabetes, osteoporosis and cancers (especially prostate cancer). We expect that producing Lycopene in cyanobacteria will enhance the efficiency of photosynthesis, therefore, leading to the increase of biofuel. Moreover, Lycopene can provide additional values in human health as we mentioned above.

　　In order to present Lycopene in Cyanobacterium Synechococcus sp. PCC7942, our chassis organism, we use the strategy－“Gene Knock Out”. Through our studies, we found that Lycopene is an intermediate in the biosynthesis pathway of beta-carotenoids in Cyanobacterium Synechococcus sp. PCC7942¹⁴ There is an enzyme, called Lycopene cyclase3, converts acyclic hydrocarbon Lycopene into the bicyclic Beta-carotene. Once we can knock out the Lycopene cyclase gene (CrtL), the Lycopene cyclase won’t exist. If the lycopene cyclase doesn’t exist, the Lycopene will remain and not be transferred into other compounds. With the accumulation of Lycopene, meeting the goal of our project is anticipated.

　　We construct a plasmid to knock out the Lycopene cyclase gene (CrtL). The plasmid contains the “upstream of CrtL” (we call it “Lycopene-US”), the downstream of CrtL(we call it “Lycopene-DS”) and Ampicillin resistant gene(AmpR). First, we use PCC7942 as our template to process polymerase chain reaction (PCR) to gain the sequence of Lycopene-US and Lycopene-DS. And we use the backbone we’ve made as the PCR template to gain AmpR sequence. Second, with the primers we’ve designed, we process a three pieces fusion PCR to connect these three sequences as the following order：Lycopene-US + AmpR + Lycopene-DS.

　　Third, the constructed plasmid containing” Lycopene-US + AmpR + Lycopene-DS” is transferred into PCC7942. There is a probability to occur Homologous Recombination¹¹ in the genome of PCC7942 through this transferred plasmid. The Lycopene cyclase gene (CrtL) will be replaced by AmpR in Homologous Recombination. Finally, the “Gene Knock Out” is completed.

　　To conform to the iGEM part registry, we use IDT to synthesis the DNA sequences of Lycopene-US and Lycopene-DS. Compared with PCR these two sequence from PCC7942 wild type, the IDT sequence won’t contain the Restriction Enzyme sites (RE site) of PstI and XbaI because we’ve changed one codon in each RE site according to the codon usage table of PCC7942. The submission parts to the iGEM Parts Registry are
　　BBa_K2350007(Lycopene-US)
　　BBa_K2350008 (Lycopene-DS).
　　BBa_K2350014 (Lycopene-US + AmpR + Lycopene-DS).

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　　We expected the transformed PCC7942 with CrtZ gene could produce zeaxanthin and become more yellow. Just like our prediction, the result showed that the transformed PCC7942 was more yellow than wild type obviously.

　　Moreover, we expected the transformed PCC7942 which produce zeaxanthin would change wavelength absorbance and have better photosynthetic efficiency.

Wavelength Absorbance

　　To test this hypothesis, we used spectrophotometer to measure the absorbance of CrtZ and wild type at 400 to 700 nm. The outcome was that the OD value of CrtZ at 400 to 500 nm was hihger than wild type. The result verified our first prediction, a change of wavelength absorbance. Not only this, the change of wavelength absorbance was at 400 to 500 nm, which was blue light. This indicated CrtZ absorbed blue light and reflected yellow light, so CrtZ was more yellow than wild type. The measurement matched what we saw intuitively.

Photosynthetic Efficiency

　　To test whether the photosynthetic efficiency of CrtZ is better than wild type, we used iodine to measure starch concentration. First, the initial concentration of microalgae of CrtZ and wild type should be same, so that the measurement could be fair. We measured the OD value at 730 nm, which represented the concentration of microalgae. Then we calculated how much microalgae and BG11 (the medium) we should add to make same amount of microalgae in each plate. Second, we started to measure starch concentration. We measured the OD value of each plate at 730 nm, which represented the cell number. Then we added 50 μl iodine into each cuvette, waited for five minutes, and measured the OD value at 620 nm, which represented the starch content. We repeated this step for seven days. Here are our results.

　　Then we divided the starch content by cell number, and we knew there was how much starch in every unit cell.

　　Then we calculated the variation of starch content per cell per day.

　　The first day, wild type (WT) had more starch than CrtZ. However, the increase of starch per day of CrtZ was more than wild type. The result implied that CrtZ could produce more starch per day than the wild type. Moreover, the result corresponds to our hypothesis -- CrtZ actually had better photosynthetic efficiency!

Survival Ability

　　We also considered about the survival ability of CrtZ. Therefore, we conducted a competitive experiment of CrtZ and wild type. We co-cultured CrtZ and wild type together, and then observed the color after seven days. Similarly, the initial concentration of microalgae of CrtZ and wild type should be same, and we used the same method we used in photosynthetic efficiency experiments.

　　After seven days, CrtZ and wild type both survived. The result indicated that CrtZ was strong and had great survival ability!

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　　pPIGBACK-CrtZ is transformed into Synechoccocus elongatus PCC7942 after 20 days cultivation, the transformants electrophoresis result is showed below. See figure 1. Transformation efficiency of pPIGBACK-CrtZ is 11.4 transformants per μg DNA, and correctness is 52% (10/19), which is relatively high compared to low successful rate of gene recombination.

　　Therefore, we can conclude that pPIGBACK is quite a reliable vector which could finish gene double-crossover homologous recombination in S. elongates PCC 7942 genome, because the successful rate of gene double-crossingover homologous recombination is low in cyanobacteria. Moreover, compare pPIGBACK-CrtZ transformants with wild type, we can assure that pPIGBACK could be express in S. elongates PCC 7942, which is such a milestone in our project. With the high correctness of pPIGBACK-CrtZ transformants, we have the confidence that multiple colors of cyanobacteria is possible and could be functional in the near future.

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1. Pei-Hong Chen, Hsien-Lin Liu, Yin-Ju Chen, Yi-Hsiang Cheng, Wei-Ling Lin, Chien-Hung Yeh and Chuan-Hsiung Chang. (2012). Enhancing CO2 bio-mitigation by genetic engineering of cyanobacteria. Energy Environ. Sci., 2012,5, 8318-8327.

2. Alexander O. Brachmann, Ferdinand Kirchner, Carsten Kegler, Sebastian C. Kinski, Imke Schmitt, and Helge B. Bode. (2011). Triggering the production of the cryptic blue pigment indigoidine from Photorhabdus luminescens. Journal of Biotechnology, 157, 96-99.

3. Xu, F., Gage, D., and Zhan, J. (2015). Efficient production of indigoidine in Escherichia coli. Journal of Industrial Microbiology & Biotechnology, 42, 1149–1155.

4. 2013 Team: Heidelberg (https://2013.igem.org/Team:Heidelberg/Project/Indigoidine)

5. Copeland,A. et al.(2014). Carotenoid pathway of Synechococcus elongates PCC 7942, complete genomegatus PCC 7942. Unpublished.

6. Shinichi Takaichi(2011). Carotenoids in Algae: Distributions, Biosyntheses and Functions. Mar Drugs. 2011; 9(6): 1101–1118.

7. Chuan-Hsiung Chang(2012). Enhancing CO2 bio-mitigation by genetic engineering of cyanobacteria. Energy Environ. Sci., 2012, 5, 8318.

8. NCBI (https://www.ncbi.nlm.nih.gov/nuccore/CP000100)

9. 2009 Team: Tokyo Tech (https://2009.igem.org/Team:Tokyo_Tech)

10. Nadja A. Henke, Sabine A. E. Heider, Petra Peters-Wendisch and Volker F. Wendisch. (2016). Production of the Marine Carotenoid Astaxanthin by Metabolically Engineered Corynebacterium glutamicum. Marine drug, 14(7): 124.

11. Chengwei Liang, Fangqing Zhao, Wei Wei, Zhangxiao Wen, Song Qin. (2006). Carotenoid Biosynthesis in Cyanobacteria: Structural and Evolutionary Scenarios Based on Comparative Genomics. Int. J. Biol. Sci., 2006, 2.

12. Miller, E. S., Mackinney, G., & Zscheile, F. P. (1935). Absorption Spectra of Alpha and Beta Carotenes And Lycopene. Plant Physiology, 10(2), 375–381.

13. David Heber, Qing-Yi Lu (2002). Overview of Mechanisms of Action of Lycopene. Experimental Biology and Medicine, 227(10), 920 – 923.

14. Cunningham, F. X., Sun, Z., Chamovitz, D., Hirschberg, J., & Gantt, E. (1994). Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942. The Plant Cell, 6(8), 1107–1121.

15. Jacobus, A. P., & Gross, J. (2015). Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli. PLoS ONE, 10(3), e0119221.

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