Part:BBa_K2350022
pPIGBACK-PrbcL-CrtZ
Part description
Zeaxanthin belongs to carotenoid family and is widely found in nature. It is also a natural yellow color making of corns, carrots and marigolds. Moreover, zeaxanthin is an essential nutrient substance to human eyes, and some healthy supplements are made of it. Most of the green plants produce zeaxanthin as an intermediate in carotenoid pathway. However, some cyanobacteria lack some genes and cannot produce zeaxanthin, such as Synechococcus elongatus PCC 7942. PCC7942 lacks only one gene making zeaxanthin, that is beta-carotene hydroxylase (CrtZ). To make Synechococcus elongatus PCC 7942 produce zeaxanthin, we constructed a plasmid BBa_K2320022 under the control of PrbcL. After the expression of CrtZ, PCC 7942 can then be yellow.
And the crtZ what we used was a part released in iGEM (BBa_I742157) .We have successfully constructed this part on our special designed backbone pPIGBACK so that it can be expressed in our microalgae and resulted in yellow microalgae.
Details
1. We studied Professor Chuan-Hsiung Chang’s paper(Energy Environ. Sci., 2012, 5, 8318: Enhancing CO2 bio-mitigation by genetic engineering of cyanobacteria) and decided to construct pigment plasmid with the same promotor. The natural ribosome binding site was also referred to it.
2. The intrinsic promoter of Rubisco large subunit (PrbcL) can overexpress foreign genes in cyanobacteria. Many plants’ proteins in photosynthesis are under regulation of PrbcL. And the high activity to express foreign genes has been proven.
3. CrtZ from Pantoea ananatis is a coding sequence of igem released part (BBa_I742157). It can lead to zeaxanthin and astaxanthin. However, the wild type Synechococcus elongatus PCC 7942 lacks it and cannot make zeaxanthin naturally.
Result
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. 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.
The right one of Figure 1 was wild type Synechococcus elongatus PCC 7942, the left one was transformant with BBa_K2320022. Obviously, the left one was more yellow than the right one. It proved that CrtZ was successfully transformed to PCC7942 and lead to zeaxanthin. See Figure 1.
Figure 1
Figure 2 is pPIGBACK-CrtZ transformants electrophoresis result. C1~C20 represent the pPIGBACK-CrtZ transformants clone 1 to clone 20, and M represents 1 Kb marker. Transformation efficiency of pPIGBACK-CrtZ is 11.4 transformants per μg DNA, and correctness is 52% (10/19), which is quite efficient because the successful rate of gene double-crossingover homologous recombination is low. See Figure 2.
Figure 2
To test whether the photosynthetic efficiency of CrtZ is better than wild type, we then used iodine to measure starch concentration. First, the initial concentration of microalgae of CrtZ and wild type should be the 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.
Figure 3 is the cell number, and Figure 4 is the starch content. See Figure 3 and Figure 4.
Figure 3
Figure 4
Figure 5 is the starch content per cell, and Figure 6 is the delta starch content compared with days. In Figure 6, the starch content changed per cell of transformants were more than wild type, and proved that photosynthesis of some transformants were comparable to or more efficient than wild type. See Figure 5 and Figure 6.
Figure 5
Figure 6
Conclusion
From the results, CrtZ was surely transformed to PCC7942 and elevated the photosynthetic efficiency of transformants.
Discussion
1.The 2011 NCTU Formosa inspired us to transform photosynthetic pigments to cyanobacteria. NCTU Formosa used zeaxanthin synthesis pathway for temperature sensory. They used E.coli and we used Synechococcus elongatus PCC7942. Furthermore, we focused on the photosynthetic efficiency.
2.There are two yellow pigments in carotenoid pathway, zeaxanthin and lutein. The reason why we did not choose lutein was that PCC7942 lacked almost every gene on α-carotene pathway. Moreover, the only gene that PCC7942 lacks on zeaxanthin pathway is an IGEM released part. So we preferred zeaxanthin to lutein.
3. We assumed that our modified cyanobacteria are able to promote photosynthetic efficiency from our modeling. Maybe they can slow down the greenhouse effect efficiently.
4.Scientists usually use knockout to change color of microalgae. For example, Kwangryul Baek blocked Chlamydomonas reinhardtii's epoxidation step from zeaxanthin to violaxanthin. They afterwards accumulated zeaxanthin in Chlamydomonas reinhardtii. However, we did not follow the mainstream method. We rather added gene to Synechococcus elongtus PCC7942.
5.Often, altering color of cyanobacteria can protract exposures to strong light, low temperatures and drought. In the future, we can test whether CrtZ transformant can bear such harsh conditions.
6.Microalgae are versatile organisms. Morning Glory Pool is one of the main attractions of Yellowstone National Park. The hot spring contains a variety of bacteria and archae that can bear different temperatures and salts. We may foresee that more microalgae will be changed to other colors for the purpose of adapting to various conditions.
Reference
1.Shinichi Takaichi(2011). Carotenoids in Algae: Distributions, Biosyntheses and Functions. Mar Drugs. 2011; 9(6): 1101–1118.
2.Chuan-Hsiung Chang(2012). Enhancing CO2 bio-mitigation by genetic engineering of cyanobacteria. Energy Environ. Sci., 2012, 5, 8318.
3.NCBI (https://www.ncbi.nlm.nih.gov/nuccore/CP000100)
4.Kwangryul Baek, Duk Hyoung Kim, Jooyeon Jeong, Sang Jun Sim, Anastasios Melis, Jin-Soo Kim, EonSeon Jin & Sangsu Bae(2016).DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Scientific Reports 6, Article number: 30620 (2016)
5.Jean-Hugues B. Hatier, Michael J. Clearwater, Kevin S. Gould(2013)The Functional Significance of Black-Pigmented Leaves: Photosynthesis, Photoprotection and Productivity in Ophiopogon planiscapus ‘Nigrescens’(https://doi.org/10.1371/journal.pone.0067850)
pPIGBACK-PrbcL-CrtZ
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- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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