Parts

New Parts

In order to increase the yield of tryptophan in E.coli, the gene of key enzyme AroG which catalizes the synthesis of DAHP by condensation of PEP and E4P, is selected to be overexpressed. To maximum the output, a mutant AroG (S211F) without Phe feedback inhibition is picked to construct the induced expression system. And this part is documented under BBa_K3832008. In addition, a toggle switch circuit with GFP and RFP is also constructed. It can achieve bistable status and switch in certain condition, like IPTG induction and 42℃ heat, and the expression of reporter mRFP and sfGFP is upregulated, resulting in toggle of fluorescence. This part is documented under BBa_K3832007. And all other new basic parts included in our project are listed in the table.

#	Name	Type	Description	Length
BBa_K3832000	aroG (Mutant S211F)	Coding	Phospho-2-dehydro-3-deoxyheptonate aldolase (mutant S211F)	1053bp
BBa_K3832003	Medium RBS (RBS-B)	RBS	A medium RBS in E.coli	10bp
BBa_K3832004	Medium RBS (RBS-D)	RBS	A medium RBS in E.coli	14bp
BBa_K3832005	Weak RBS (RBS-H)	RBS	A medium RBS in E.coli	11bp
BBa_K3832007	Toggle-switch circuit with GFP and RFP	Composite	A bistable toggle switch with two promoter-repressor systems (lacUV5 promoter-LacI and lambda-CI857) and reporter genes (sfGFP and mRFP)	3685bp
BBa_K3832008	Induced expression circuit of aroG (Mutant s211f)	Composite	An expression system of aroG-S211F induced by IPTG	2503bp

Part improvement

1. Introduction

AroG (3-deoxy-7-phosphoheptulonate synthase, EC 2.5.1.54) is a key enzyme in the metabolism of aromatic amino acids, catalysis following reaction:
phosphoenolpyruvate + D-erythrose-4-phosphate+H2O = 3-deoxy-D-arabino-hept-2-ulosonate-7-phosphate +phosphate

Wild aroG functions in the form of tetramer, which can be inhibited by its allosteric inhibitors Phenylalanine (Phe). In order to increase the production of downstream products such as tryptophan, Ser on site 211 of aroG was mutated to Phe to remove the inhibitory effect of Phe. The CDS of the mutant (aroG-S211F, Part:BBa_3832000) is our improvement version of Part:BBa_K1060000 which encodes wild-type aroG.

We used protein structure prediction tool (Alphafold2) to predict the structure of the mutant aroG-S211F, and compared its ability to bind to substrates in the presence of Phe with that of the wild type.

In our project, aroG-S211F is used to improve the production of tryptophan. Considering the over-expression of aroG-S211F could significantly reduce the amount of substrate (glucose) entering the glycolysis reaction, in turn affects the normal process of cell proliferation, the expression of aroG is designed under strict control by toggle-switch circuit.

2. Construction

An inducible circuit (Part:BBa_K3832008) is constructed to characterize and measure the function of aroG-S211F (Fig.2.1). LacUV5 promoter is used while LacI is also contained in our circuit as a repressor.

Fig. 2.1 Design of the inducible circuit for aroG-S211F

Both GolgenGate assembly and In-Fusion assembly are used to construct the circuit from basic parts and insert into pET28a+ vector (In-Fusion assembly is done with the help of partner team NWU-CHINA-A as our collaboration).

Reaction:
Insert (purified PCR product)/ng= Length/bp × 1.08×102 Vector (purified PCR product)/ng= Length/bp × 2.16×102 BsaI-HFv2 (20U/ul) = 1 ul T4 ligase (1000U/ul) = 1 ul T4 ligase buffer (10×) = 2 ul ddH20 = 20 ul

Condition:

Temperature	Time	Cycle
37 ℃	15 min	10
16 ℃	10 min	10
37 ℃	10 min	1
65 ℃	10 min	1
80 ℃	10 min	1
Store at 4 ℃

Transformation:
Using TreliefTM 5α Chemically Competent Cell (Tsingke Biotechnology Co., Ltd.) Add 20 ul assembly product in 50 ul competent cell, ice bath for 30 min. Heat shock 42℃ for 45 sec. Ice bath for 2 min. Recover in 1ml SOC medium, 37℃, 200rpm, for 1 hour. Spread to LB plate with 50 ug/ml kanamycin, 37℃ for 10-16 hours.

Fig.2.2 and Fig.2.3 respectively show the fragment of the inducible circuit and corresponding cloned vector.

3. Measurement

3.1 RT-qPCR

RT-qPCR is used to detect the transcription of aroG-S211F as first step.

Cultivation: Using LB liquid medium, 37℃, 200rpm.
Inducing condition: 1 mM IPTG, over 8 hours.
Total RNA extraction: Using RNAsimple Total RNA Kit,DP419 (TIANGEN BIOTECH (BEIJING) CO.,LTD.)
cDNA preparation: Using Evo M-MLV RT Mix (Vazyme Biotech Co.,Ltd); template concentration: 50ng RNA/ul; reaction condition: 37℃ 15min, 85℃ 15sec.
qPCR: Using ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co.,Ltd).
Relative Normalized Expression data is calculated by using the equation below, Relative Expression = 2-[ΔCt(T)-ΔCt(C)] where ΔCt(T) represents the difference between Ct value of target gene and internal standard gene in treatment group; ΔCt(C) represents the difference between Ct value of target gene and internal standard gene in negative control group.

Result:
As in Fig.3.1, expression of aroG-S211F under induction by IPTG is higher than that in un-induced group and negative control (DH5alpha with blank pET28a+ vector).

Fig.3.1 The relative mRNA level of aroG-S211F in DH5alpha strain with Part:BBa_K3832008 inserted in pET28a+ vector.

3.2 Characterization

3.3.1 Growth Curve

As is showed in Fig.3.2, by using the Logistic equation to fit the growth curve, the inhibitory effect of aroG expression on cell proliferation was verified (as the parameter r decreased in E.coli with aroG-S211F and induced by IPTG, which represents the reciprocal of the time it takes for the population to double).
Cultivation condition: 20 ml LB medium, 37℃, 200rpm; Inoculation dose: 20 ul (0.1%)

Fig. 3.2 (a) The population density of E.coli was measured at 600 nm by colorimetry. The scatter represents the result of the measurement. The Logistic equation was used to fit the growth curve, and the fitting results were shown in the curve. (b) and (c) respectively show the growth parameters K (environmental capacity) and r (intrinsic growth rate) of different experimental groups obtained from the fitting results in (a).

3.3.2 Tryptophan Production

Fig. 3.3 shows the yield of tryptophan. The engineered E.coli with aroG-S211F induced by 1 mM IPTG resents an increased production of tryptophan, comparing in absent of IPTG or aroG-S211F (within blank pET28a+ vector).

Concentration of tryptophan is calculated by standard curve in Fig.3.4. Tryptophan production is calculated by removing the concentration of blank medium, and normalized by divided by OD600 (reflecting cell density).

Fig. 3.1 The relationship between tryptophan concentration in culture medium and culture time. The concentration of tryptophan is measured by PDAB chromogenic method.

Freeze-thaw bacterial culture medium with suspension cells for over 3 times.
Add 100 ul medium into 400 ul PDAB (p-dimethylaminobezaldehyde) solution (3 mg/ml in 9 M solution of sulfuric acid). Then keep at 60℃ for 20 min.
Add 3 ul 0.5% (w/w) solution of sodium nitrite. Then keep at 60℃ for 15min.
Measure absorption under 590 nm wavelength (OD590).

The same method should be used to determine the OD590 of a tryptophan standard solution at a known concentration to obtain a standard curve. Standard curve measured and used in our experiment is as Fig.3.4.

Fig.3.4 Standard curve of measuring tryptophan tryptophan by PDAB method.

Team:XJTU-China/Parts