Team:BUCT/Parts

Parts
Overview
In the part of producing GABA, we redesigned the gadB of 2019_SCUT (BBa_K3100017) and characterized its the activity of GadB in different pH value. We have also characterized our new part, gadB-E89Q+z452-466 (BBa_K3875000) from E.coli. The results of the two parts are compared. It is indicated that GabB transcribed by our mutated gene (gadB-E89Q+z452-466) is active in a wide range of pH, while GadB transcribe by wild gene is only active at pH 4.6.
gadB(mut)
There are many E.coli glutamate decarboxylase isoforms, and GadB is one of them. Glucose and Acetyl-CoA can be consumed in the citric acid cycle to form ɑ-ketoglutaricacid, which can produce L-glutamate under the catalyst of gdhA, L-glutamate can be future turned into GABA(4-aminobutyricacid) under the catalyst of L-glutamate decarboxylase (GadB) [1]. Concerning that the wild-type GadB has an optimal activity at pH 4.5, which is not a suitable condition for cell growth, we want to mutate gadB to a mutant gene (gadB E89Q Δ452-466). Then the GadB transcribed by our gene gadB(mut) will be active in a wide range of pH, so as to overproduce GABA [2].
Characterization
we linked the sequence of fadD (BBa_K3040118) and fadL (BBa_K3040117) into pCS27 backbone, which contains lac operator so that FadD and FadL can be induced by IPTG. The plasmids were tranferred into E.coli 1917 Nissle strain and we induced the recombinant overnight. The expression of the fusion protein was determined by SDS-PAGE.
Also, this year the BUCT team chose MazEF TA system as a suicide element, presenting "basic" and "oxygen-altering death" circuits that act as a kill switch for E. coli.

Express antitoxin mazE (BBa_K302032)under the control of the phyb promoter, which is only on under microaerophilic conditions.

The expression of the toxin mazF (BBa_K823044) is induced by dietary lactose. Under microaerophilic conditions in the human intestine, engineered bacteria will survive by expressing the antitoxin MazE. However, once bacteria are excreted to the aerobic environment, MazE will no longer be produced, causing bacteria to commit suicide.

To test the function of the toxin, mazF will be controlled under an inducible promoter.
Improved Part
In improvement, we mutated the gene gadB of 2019_SCUT (BBa_K3100017) and tried to characterize GadB and GadB(mut) in different pH value. Then results of the two parts are compared.
Also, this time we construct a plasmid (BBa_K3875007) to produce GABA with the consumption of the simple carbon sources. By doing this, we supplement all paths of producing GABA from simple carbon sources to the production GABA. This is an improvement for (BBa_K2326005), which just produced GABA from middle step.
Basic parts

Basic parts

Name

Type

Description

Designer

Length

BBa_K3875000

Coding

gadB-E89Q+△452-466

Yimiao Lin

1356

BBa_K3875001

Coding

AntrpC

Yimiao Lin

2310

BBa_K3875003

Coding

trpG

Yimiao Lin

615

BBa_K3875004

Coding

p4h(W179F)

Yimiao Lin

891

BBa_K3875005

Coding

pcd

Yimiao Lin

357

BBa_K3875008

Coding

gdhA

Yimiao Lin

1344

BBa_K3875009

Coding

trpE

Yimiao Lin

1563

BBa_K3875015

Coding

AmpR

Yimiao Lin

861

BBa_K3875016

Coding

pUC18-LYF

Yimiao Lin

934

BBa_K3875018

Teminator

Teminator

Yimiao Lin

32

BBa_K3875019

Regulatory

J23119(SpeI) promoter

Yimiao Lin

35

BBa_K3875020

RNA

gRNA trageting fadR

Yimiao Lin

20

BBa_K3875021

Conjugatiom

Cas9 binding

Yimiao Lin

34

BBa_K3875023

RNA

sgRNA-R

Yimiao Lin

25

BBa_K3875026

Composite

Lac-mazF-T1-lac-phyb-mazE-T1

Yimiao Lin

1064

BBa_K3875027

Composite

Lac-mazF-T1-lac-vgb-mazE-T1

Yimiao Lin

1005

BBa_K3875028

RBS

aggaga

Yimiao Lin

6

BBa_K3875029

RBS

aacaattgaaattattcctc

Yimiao Lin

21

Composite parts

Composite parts

Name

Type

Description

Designer

Length

BBa_K3875001

Composite

Lac-fadL-fadD-T1

Yimiao Lin

3412

BBa_K3875006

Composite

Lac-trpE-trpG-AntrpC-T1

Yimiao Lin

4672

BBa_K3875007

Composite

Lac-gadB(mut)-gdhA-T1

Yimiao Lin

2835

BBa_K3875010

Composite

Lac-trpB-p4h-pcd-T1

Yimiao Lin

2603

BBa_K3875011

Composite

Lac-trpE-trpG-AntrpC-T1-lac-trpB-p4h-pcd-T1

Yimiao Lin

7266

BBa_K3875013

Composite

Lac-mazF-T1

Yimiao Lin

486

BBa_K3875014

Composite

T7-gaB(mut)

Yimiao Lin

1385

BBa_K3875017

Composite

AmpR promoter-AmpR-pUC18-LYF-Teminator

Yimiao Lin

1956

BBa_K3875022

Composite

J23119 promoter-gRNA targeting fadR-Cas9 binding-Teminsator

Yimiao Lin

145

BBa_K3875030

Composite

Phyb-mazE-T1

Yimiao Lin

530

Improved parts

Improved parts

Part Number

Type

Description

New Part Number

New Description

BBa_K3100017

Coding

Wild gadB

BBa_K3875000

gadB-E89Q+△452-466

BBa_K2326005

Coding

LacI - pTac - gadA + 6xHis Tag - Terminator

 

BBa_K3875007

Lac-gadB(mut)-gdhA-T1

Characterized parts

Characterized parts

Part Number

Type

Description

BBa_K3040118

Coding

fadD

BBa_K3040117

Coding

fadL

BBa_K823044

Coding

mazF

BBa_K302032

Coding

mazE



Reference

[1] Chae, T. U., Ko, Y. S., Hwang, K. S., & Lee, S. Y. (2017). Metabolic engineering of Escherichia coli for the production of four-, five- and six-carbon lactams. Metabolic engineering, 41, 82–91. https://doi.org/10.1016/j.ymben.2017.04.001

[2] Sheng, L., Shen, D., Yang, W., Zhang, M., Zeng, Y., Xu, J., Deng, X., & Cheng, Y. (2017). GABA Pathway Rate-Limit Citrate Degradation in Postharvest Citrus Fruit Evidence from HB Pumelo (Citrus grandis) × Fairchild (Citrus reticulata) Hybrid Population. Journal of agricultural and food chemistry, 65(8), 1669–1676. https://doi.org/10.1021/acs.jafc.6b05237

The whole BUCT team would like to thank our sponsors. Especially:
                               
Tel: 86+18718806286
Address: No.15 North Third Ring East Road,Chaoyang District,Beijing,P.R.China. Beijing University of Chemical Technology

BUCT--iGEM
BUCT--iGEM