Part:BBa_K4171023

Ptrc-gadB-B0015

Backgrounds

This part is for GABA biosynthesis. pSB4KI-P_trc-gadB-B0015 was constructed to express glutamate decarboxylase (GAD), an enzyme responsible for γ-Aminobutyric acid (GABA) production.

Usage

This part is used to produce GAD, an enzyme that catalyzes the reaction of glutamate or MSG to GABA. In our project, we used GAD to produce GABA as a demonstration of crucial substance production in our engineered bacteria.

Fig. 1. GABA synthesis pathway

Characterization

The glutamate decarboxylase gene gadB (BBa_M50089) was synthesized and placed under the control of the P_trc and double terminator B0015 in vector pSB4KI. The plasmid containing P_trc, terminator B0015 and melA gene was transformed into E. coli DH5a and completed the construction.

Fig. 2. Confirmation of pSB4KI-P_trc-gadB-B0015 (BBa_K4171023) by double digestion. M: Marker; Lane 1: pSB4KI-P_trc-gadB-B0015 (6370 bp); Lane 2: pSB4KI-P_trc-gadB-B0015 without digestion (negative control).

Below is the GABA production result of culturing the bacteria with glutamic acid or MSG as a substrate. GABA production did not show a significant difference between adding glutamic acid and MSG. Since MSG is much cheaper than glutamic acid, it is suggested to convert glutamic acid into MSG in the industry.

Fig. 3. GABA production under different substrates (Glutamic acid and MSG).

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 739
Illegal EcoRI site found at 1177
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 739
Illegal EcoRI site found at 1177
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 739
Illegal EcoRI site found at 1177
Illegal BamHI site found at 654
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 739
Illegal EcoRI site found at 1177
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 739
Illegal EcoRI site found at 1177
Illegal NgoMIV site found at 1305
Illegal AgeI site found at 567
1000
COMPATIBLE WITH RFC[1000]