Plasmid Construction And Expression
We constructed three basic expression plasmids to produce Tyrian Purple.(Click here to know more). After transferring them into E.coli BL21(DE3), we measured the growth curves of each strain (Fig 1. a,b,c).
Fig.1 a. FLS growth curve. b. TnaA growth curve. c. MaFMO growth curve.
As we can see, all of these bacterial growth curves experienced lag phase, logarithmic phase, stationary phase and decline phase. From this, we obtained the best induction time of these strains.
Expression of Fre-Linker-SttH, TnaA, MaFMO
For FLS，some references and our pre-experiment show that it will express as inclusion body at high temperature (37℃). So, to obtain the best induction condition, we induced FLS at different temperatures and time (Fig.2). Then, we induced TnaA and MaFMO at different time and temperatures respectively (Fig.3, Fig.4).
Fig.2 SDS-PAGE of FLS expression. Induce condition: LB media, OD600 0.6 ~ 0.8, IPTG 0.1 mM. 1:negitive control; 2: 9h 37℃; 3:9h 30℃; 4:9h 23℃; 5:9h 17℃; 6:15h 37℃; 7:15h 30℃; 8:15h 23℃; 9: 15h 17℃.
Fig.3 SDS-PAGE of TnaA expression. Induce condition: LB media, OD600 0.6 ~ 0.8, IPTG 0.1 mM, 37 °C and 200 rpm.1:negitive control; 2: 9h; 3:10.5h; 4:12h; 5:13.5h; 6:15h; 7:16.5h; 8: 18h; 9: 19.5h.
Fig.4 SDS-PAGE of TnaA expression. Induce condition: LB media, OD600 0.6 ~ 0.8, IPTG 0.1 mM, 18 °C and 200 rpm. 0:negitive control; 1:4h; 2:5h; 3:6h; 4:7h; 5:8h; 6:9h; 7:10h; 8:11h.
Tyrian Purple Production
Whole-cell reactions
Limited by Fre-Linker-SttH expression, we decided to use 6-Br-Trp as the substrate to go on the whole-cell reactions. First of all, we expressed TnaA (30℃,16h) and MaFmo (26℃,4h) with 6-Br-Trp containing LB medium and common LB medium respectively. The cells were resuspended in 50mM NPB solution after centrifugating through high-speed freezing centrifuge. Then the TnaA and MaFMO suspension were mixed and incubated in 30℃, 200rpm for 12h. In this way, we successfully obtained the target product Tyrian Purple (Figure.5 a,b). At the same time, we also obtained the optimal mixing ratio of TnaA to MaFmo by comparative experiments. Additionally, we found that the NPB buffer was more effective than the PBS buffer, because Na+ was able to assist transmembrane transport of 6-Br-Trp and 6-Br-indole (Fig.5 e).
Fig.5 a.TnaA:MaFMO (1.2:1), LB medium resuspended. b. TnaA:MaFMO (1.2:1), NPB resuspended. c. TnaA:MaFMO(1:1), NPB resuspended. d.TnaA:MaFMO (1.2:1), NPB resuspended. e. TnaA:MaFMO(1:1), PBS resuspended.)
Lyophilized Powder
Tyrian Purple contained bacterial fluid was frozen at-80℃for 12h, and then made into lyophilized powder by a freeze dryer.
The Improvement Production
The effect of Fre
SttH was successfully overexpressed in E. coli, but the enzyme mostly existed in an insoluble form (Fig.6). To test Fre can improve the bromination reaction rate, we did a comparative experiment between SttH and Fre-Linker-SttH. First ,we did PCR with the primer of SttH, it showed good results.
Fig.6 Agarose-gel electrophoresis of SttH.
Then ,we connected SttH to pET-28a(+), transfered into BL21(DE3) and added IPTG to induce SttH expression.
Improvement through In-Fusion
To achieve the highest yield of producing and simply the reaction process, we made TnaA and MaFMO in one plasmid use In-Fusion method. After PCR reaction, DNA electrophoresis bands were shown below.
The pET-28a(+) expression vector was chosen, BamHI and Hind III restriction sites were selected.(click here to learn more protocol)
Fig.7 1：PCR results of TnaA; 2: negitive control; 3: PCR resulting MaFMO; 4: linear pET-28a(+) after enzyme digestion.
Fig.8 1:negitive control; 2: 3.5h; 3:9h; 4:10h; 5:11h; 6:12h;
We also did a whole cell reaction, this improvement saved us nearly twice as long(Fig.9,10). We suspected that this is due to the transmenmbrane transport of 6-Br-indole was skipped.
Fig.10 The absorption curves of Tyrian Purple production.
Fig.11 In-Fusion Production.
CRISPR Cas9
Since TnaA is an endogenous enzyme of E.coli, when the strain with engineered TnaA, there will be a competition between TnaA and SttH, and produce indindex (indole). So, we imported the Cas9 plasmid and the Target plasmid into BL21 (Fig.12), and designed primers which contained the upstream and downstream sequence of the E. coli genome TnaA, then we evaluated PCR products by agarose gel electrophoresis (Fig.13) to ensure that we successfully knocked down the TnaA.
Fig.12 BL21 with Cas9 and Target.
Fig.13. PCR products using the upstream and downstream sequence contained primers.
After knockdown of TnaA, we eliminated CRISPR Cas9 plasmids in E.coli BL21 (DE3) and grown them in Kan and Amp contained LB medium for ensurance (Fig.14). Subsequently, we transformed FLS and TnaA-MaFMO plasmids into E.coli BL21 (DE3) TnaA(-) for protein expression.
Fig.14
Dyeing Effect
After obtaining Tyrian Purple, we immediately performed staining experiments on different materials. According to the results, the wool and silk showed the best effect (Fig.15), and common materials can be well colored.
