Team:NYMU-Taipei/Pigments
Pigments
In our project, we transfer five types of pigment-related gene sequence (Indigoidine, Zeaxanthin, Melanin, Astaxanthin and Lycopene) into our cyanobacteria. We expect to get five different colors of microalgae, so we could see whether changing the original color of microalgae would change wavelength absorbance and have better photosynthetic efficiencies. Due to better photosynthetic efficiencies, we could elevate oil accumulation in microalgae, which would have great benefit in both industry and scientific usage.
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.
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.
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.
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).
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 higher 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!
pPIGBACK-CrtZ is transformed into Synechoccocus elongatus PCC7942 after 20 days cultivation, the transformants electrophoresis result is showed below. 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.
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.
