Part:BBa_K2947001
UGT33
UDP-glucose-4-O-glucosyltransferase(T8GT) protein coding region.UGT33 is the most active gene among R. rosea Tyrosol-Modifying UGTs. The protein it encodes reduces tyrosol to produce salidroside. UGT33 is used as the last gene to complete salidroside biosynthesis from tyrosine.
Usage and Biology
The UGT super-family is one of the largest enzyme families in the plant kingdom. Previous research has shown that(Michel et al., 2017) among all the genes encoding UDP-glucose-4-O-glucosyltransferase(T8GT), UGT33 is the most active T8GT.
When 2019 GDSYZX is trying to transfer UGT33 to vector, the transfer of UGT33 into carrier was not smooth during the experiment. We started with the traditional enzyme training but it didn't work. Then we switched to a ligase, and it still was a false positive. Finally, we used the recombination link approach and successfully transferred UGT33 into the plasmid.
The protoplast and salidroside standard samples of arabidopsis thaliana transfected with UGT33 and 4HPAAS genes were compared by liquid chromatography. The peak value of the product measured in sample No. 1 was basically consistent with the peak value of salidroside standard samples.
Therefore, we can determine that the transfected protoplast of arabidopsis thaliana can produce salidroside.
MODELING(with 4HPAAS:BBa_K2947000)
By Using Image J software to analyze the relative brightness of protein bands, we obtained the data of gray value of two genes/4h varying with time. There is a positive correlation between gray value / 4H and protein expression, so we use the standardized gray value / 4H to reflect protein expression. Then we use SPSS software to draw the scatter plot of gray value / 4H about time to judge the overall trend. Among them, we changed the SPSS output format to English. (The relative gray value is equal to the following gray value divided by the gray value of 4h)
We found that these scatters showed a quadratic function parabola trend, so we square the time data. Next, we test Pearson correlation coefficient of time, time squared and gray value / 4H to check whether there is a linear correlation between them.
Then we found that the correlation between UGT33 gene variables was quite high, P value was less than 0.05, with a high significance. The correlation coefficient of 4HPAAS gene was higher than 0.6, P value was higher than 0.05, but less than 0.2. Although the degree was not significant, the correlation coefficient was higher. It can be concluded that the binary linear regression equation can be constructed between the relative gray value of two gene sequences and the square term of time . By using the least square method, we get the regression equation between relative gray value and time.
Relative gray value = beta0 + beta1 * time + beta2 * time squared
New documentation edited by SYSU-CHINA 2022 on 6th Oct 2022---------------------------------------------
It is also feasible to express the UGT33 in E.coli instead of Arabidopsis thaliana. We find some articles about the related method and experimental results of HPLC, which may be useful for future iGEMers to conduct the related experiments. We also document our experiment below for reference.
1 Method
To analyze the formation of tyrosol, hydroxytyrosol, and salidroside by HPLC, the mobile phase was composed of water (solution A) and acetonitrile (solution B), which were combined with 0.1% formic acid. The elution program was as follows: the proportion of solution B was gradually increased from 10% to 40% over 8min, increased to 90% over 4min, and then maintained over 3min. Finally, the proportion of solution B was rapidly decreased to 10% and maintained at that composition for 5min. The flow rate was 1ml/min, the HPLC result of salidroside is as follow.
The mass spectrometry parameter of salidroside: 1 H NMR (400 MHz, DMSO-d6) δ 7.04 (d, J = 8.3 Hz, 2 H), 6.67 (d, J = 8.3 Hz, 2 H), 4.17 (d, J=8.5Hz, 1H), 3.87 (dd, J=16.0, 8.6Hz, 1H), 3.67 (d, J=11.4Hz, 1H), 3.56 (dd, J=16.0, 8.8Hz, 1H), 3.44 (dd, J=11.4, 5.4Hz, 1H), 3.15 (t, J=8.5Hz, 1H), 3.07 (m, 1H), 3.06 (d, J=8.5Hz, 1H), 2.96 (t, J=8.5Hz, 1H), 2.73 (m, 2H); 13C NMR (100MHz, DMSO-d6) δ 155.6, 129.7, 128.6, 115.0, 102.8, 76.8, 76.7, 73.4, 70.0, 69.9, 61.0, 34.8. The mass spectrometry is as follow
2 Experiment
Based on the results of the catalytic experiments, we selected UGT33 which is the enzyme with the highest catalytic efficiency. further, we wanted to obtain higher yields of salidroside by changing the freeze-thaw conditions. Thus, we set the thawing temperature gradients ( 42C˚, 47C˚, 52C˚, 57C˚,62C˚), we record the experimental result below.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 624
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 34
Illegal SapI site found at 460
| None |