Part:BBa_K3360012

tac-rbs-bglA-T

This part contains the basic parts that can efficiently express the β-glucosidase A of Thermotoga maritima in E. coli.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1603
  Illegal AgeI site found at 953
  
• 1000
  COMPATIBLE WITH RFC[1000]

SZU-China 2020 TEAM

Beta-glucosidase is a cellulase enzyme, which can catalyze the reaction of geniposide with amino acids to produce gardenia blue pigment. In order to find an enzyme that has good tolerance to the extreme industrial environment and can achieve rapid color development in laboratory conditions, we finally created an extremely heat-resistant β-glucosidase derived from Thermotoga marina.

We chose the plasmid pGEX-4T-1-H, and added a GST tag to it and optimized its gene sequence for E. coli codons. Under IPTG induction, the enzyme can be expressed efficiently.

Fig.1 beta-glucosidase Plasmid vector diagram

We synthesized the above-mentioned vector and double-enzyme digested it. After DNA electrophoresis, the following map was obtained. The picture shows that after double enzyme digestion, the band 1 is lower than the band 2, and the size is about 5000bp, which is the same as our prediction, indicating that the gene transformation is successful.

Fig.2 Agarose-gel electrophoresis of beta-glucosidase

Next, we did the polyacrylamide gel electrophoresis on these protein expressions and observed obvious protein expressions as shown in the figure. The induced group 1 marked with a rectangular white box is our induced beta-glucosidase.

Fig.3 Protein electrophoresis of beta-glucosidase

We constructed a recombinant plasmid pGEX-4T-1-H-beta-glucosidase with GST and transformed it into E.coli BL2l. After culture in a shaker at 37°C, bacterial were collected after IPTG expression was induced, and beta-glucosidase was collected after breaking. Before the specific measurement of enzyme activity, we drew the standard curve of beta-glucosidase enzyme activity.

Here, we define the unit enzyme activity per unit volume of beta-glucosidase (U/ml) as the amount of 1nmol p-nitrophenol produced per unit volume per minute.

We verified its enzyme activity temperature. It can be seen that it has the best enzyme activity at 90°C, which is the same as described in the literature. We also set a time gradient to detect the relationship between enzyme activity and reaction time at 80℃， and got the enzyme activity-time curve as shown in the figure. It can be seen that the enzymatic reaction rate becomes slower and slower as time flows by.

Fig.4 (a) standard curve of bata-glucosidase enzyme activity (b) beta-glucosidase enzyme activity-temperature curve (c) beta-glucosidase enzyme activity-time curve

Then we follow our best formula: add 440μl geniposide, 6μl glycine, 50μl beta glucosidase enzyme solution, and 504μl PBS buffer to the 1ml reaction system. And we prepared 250ml gardenia blue pigment solution. The color and output are within our expectations.

Fig.5 Homemade Gardenia Blue Solution