Part:BBa_K5226087
PR/PRM -tnaA -fmo
Contents
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1960
Illegal PstI site found at 724 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1960
Illegal PstI site found at 724 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1960
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1960
Illegal PstI site found at 724 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1960
Illegal PstI site found at 724
Illegal NgoMIV site found at 1912
Illegal AgeI site found at 1347
Illegal AgeI site found at 2056
Illegal AgeI site found at 2386 - 1000COMPATIBLE WITH RFC[1000]
Introduction
One of the goals of iGEM SCUT-China-A is to use synthetic biology tools to obtain Halomonas strains that can produce tyrian purple. We chose to introduce four enzymes that is either necessary or beneficial to the production of tyrian purple. There were stth,fre,tnaA and fmo. Because both Stth and TnaA can utilize tryptophan, and tryptophan has a stronger preference for TnaA than for Stth, we introduced the thermalsensitive bio-switch that we built for Halomonas TD to separate the expression of the two enzymes to increase yield.
Usage and Biology
This is a composite part used to convert 6-Br-Trp to 6-Br-indole and further to Tyrian Purple. TnaA is a kind of tryptophanase and this protein catalyzes the conversion of 6-Br-Trp into 6-bromoindole (6-Br-indole). MaFMO is a kind of flavin-containing monooxygenase and the protein catalyzes the conversion 6-Br-indole into 6,6'-dibromoindigo (6BrIG, also known as Tyrian purple). They are fused together with the common rigid linker EAAAKEAAAK. Through introducing thermalsensitive bio-switch into the synthesis pathway of Tyrian purple, we could use temperature to separate the expression of stth and tnaA, thus improve the production of 6-Br-Trp and then the Tyrian purple. Considering its importance and expression intensity, we selected the Mmp1 inducible promoter and set a series of IPTG concentrations during fermentation to induce the most suitable expression intensity for this step.
Experimental characterisation
growth conditions
Fermentation
Strain preparation
experimental design
Other variables were the amount of IPTG and tryptophan added. We set two gradients for each of the two variables: IPTG(mg/L)=5,20 and tryptophan(g/L)=0.4,0.8.
At 16,24,32h, we took a batch of samples and switched the temperature to 37℃, in case to find a better timing to switch temperature through the yield of 6-Br-Trp.
Strain1, 3 and 4 was for shake flask fermentation to test the production of Tyrian purple. Strain 2 was for deepwell plate fermentation to test the leakage of our T-switch in the synthesis of Tyrian purple as a control group.
Post fermentation treatment
(1) 6-Br-Trp
The LC conditions were as follows: solvent A was Water with 0.1% formic acid; solvent B was Acetonitrile; flow rate 0.4 mL/min; gradient ramp held 5% B for 2 min, ramped to 20% B from 2 to 5 min, ramped to 95% B from 5 to 12 min, held at 95% B until 16 min, then re-equilibrated at 5% B from 16.1 to 20 min. LC/MS data were collected using Agilent MassHunter Workstation LC/MS Data Acquisition for 6500 series Q-TOF (Version 10.1) and analyzed using Agilent MassHunter Workstation Qualitative Analysis (Version 10.0) software.
（2）Tyrian purple
Two solvent reservoirs, containing (A) water + 0.1% (v/v) TFA and (B) acetonitrile + 0.1% (v/v) TFA, were used to separate the Tyrian purple components, under gradient elution. The gradient elution program is summarized as below. Separation was performed with a flow rate of 0.5 mL min(−1) and the analytes were eluted in approximately 20 min.The final steps, up to 30 min, were included to reach the initial conditions and achieve mobile phase stabilization. Peak purity control and identification of the peaks in standard solutions and samples was performed by means of the diode array detector. The analytes were quantified at 288, 299, 540 and 590 nm.
Gradient elution program (flow rate 0.5 mL/min):
(3)Fluorescence Characterisation
-Add 1ml 60LB, 0.1% chloramphenicol and spectinomycin to the well, pick up the strain and add it to the hole, and then put the deepwell plate in the shaker at the corresponding temperature for 12-14 hours. Usually, for each strain we would prepare two wells in case of emergency.
-Add 1ml 60LB, 10μl bacterial fluid of last step,0.1% chloramphenicol and spectinomycin to the well, then put the deepwell plate in the shaker at the corresponding temperature for 12 hours.
-Take 100μl bacterial fluid of last step, add 400μl deionised water to the bacterial fluid, then centrifuge for 5 minutes, discard the supernatant and add 500μl 1X PBS, take 200μl into the enzyme standard plate to measure fluorescence using microplate reader（absorbance 600, excitation/emission wavelength 488/520）
Data Processing and Analysis
We tested samples taken from group 1 and group 3 as experimental group and control group. The result showed that the experimental group produced 75.50 mg/L and 165.36 mg/L of tyrian purple under the condition of switching to 37°C to continue culture after 36 hours of cultivation at 30°C and keeping in 30°C in the whole process respectively.
The ratio of Tyrian purple to indigo yield expressed under the same switching temperature and Trp,IPTG protocol is as follows.
At the same time, we tested the fluorescence intensity(FI) of group 2 to see the expression intensity of pR/pRM-tnaA-fmo in different timing. The result showed that most of the fluorescence leakage were controlled below 20, and a small number of anomalous data are believed to be caused by the poor uniformity of the shaker, which led to the lack of strict temperature control. Fermentation results from deep-well plates showed that the separation of Stth and TnaA expression using our T-switch was effective.
References
[1]Lee, J., Kim, J., Song, J. E., Song, W.-S., Kim, E.-J., Kim, Y.-G., Jeong, H.-J., Kim, H. R., Choi, K.-Y., & Kim, B.-G. (2021). Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nature Chemical Biology, 17(1), 104–112. [2] Zeng, J., Zhan, J. Characterization of a tryptophan 6-halogenase from Streptomyces toxytricini . Biotechnol Lett 33, 1607–1613 (2011). [3]Athina Vasileiadou, Ioannis Karapanagiotis, Anastasia Zotou, Determination of Tyrian purple by high performance liquid chromatography with diode array detection,Journal of Chromatography A,Volume 1448,2016,Pages 67-72,https://doi.org/10.1016/j.chroma.2016.04.046.
| None |