Team:NYMU-Taipei/Composite Part
Composite Part
BBa_K2350022
Part Description
Zeaxanthin belongs to the carotenoid family and is widely found in 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 health supplements are made of it. Most of the green plants produce zeaxanthin as an intermediate in the 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 construct 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 use was a part released in iGEM (BBa_I742158).We have successfully constructed this part on our special design backbone pPIGBACK so that it can express in our microalgae and result 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 promoter. The natural ribosome binding site is also referred to it.
2. The intrinsic promoter of Rubisco large subunit (PrbcL) can overexpress foreign genes in the cyanobacteria. Many plants’ proteins in photosynthesis are under regulation of PrbcL. And the high activity to express foreign genes has been proved.
3. CrtZ from Pantoea ananatis is a coding sequence of igem released part (BBa_I742158). It can lead to zeaxanthin and astaxanthin. However, the wild-type Synechococcus elongatus PCC 7942 lacks it and cannot make zeaxanthin naturally.
Result
Figure 1 shows that the transformed PCC7942 with CrtZ gene is more yellow than wild type
Figure 2 is pPIGBACK-CrtZ transformants electrophoresis result. C1~C20 represents the pPIGBACK-CrtZ transformant clone 1 to clone 20, and M represnets 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.
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 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 the same amount of microalgae on 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 cell number.
Figure 4 is the starch content.
Figure 5 is starch content per cell, and Figure 6 is the variation of starch content per cell per days. In Figure 6, the starch content change per cell of transformants are 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 is starch content per cell.
Figure 6 is the variation of starch content per cell per days.
