Team:BUCT-China/Engineering

Step1
Step2
Step3
Step4
Reference

Engineering Success

Due to the coronavirus in 2021, we don’t have sufficient time to enter the laboratory to complete the experiment. We completed the experimental design of this project in the early stage and completed part of the experiments in the limited time. We hope to verify the feasibility of our experimental design through part of the experimental content, and do a good foundation for our follow-up work. This page contains the results of our experiments regarding to first section.

We have described our project design ideas (including project section I and section II, design ideas, 4 steps in section I , etc.) in the design page of Wiki：
https://2021.igem.org/Team:BUCT-China/Design

Step1

We take advantage of the fatty acid synthesis pathway of E.coli to synthesize myristoleic acid from glucose, we construct an artificial metabolic pathway that can desaturate the ACP-fatty acid by Δ9 desaturase(TsAab2) and finish the fatty acid synthesis to produce 14C fatty acid by thioesterase (UcFadD).

T: Tsfab2 desaturase gene (promoter ARA)
U: Ucfadd thioesterase gene (promoter LAC)

Plasmid pet28a-tsfab2-ucfadd was designed. The T (desaturase gene) gene fragment was connected with arabinose promoter C to obtain C promoter Ara. Then, the U (thioesterase gene) fragment was connected to the t-weight long fragment through T4, and the T-U recombinant plasmid was constructed as the target plasmid.

For the pET28a-TsAab2-UcFadD we want to build, we first construct plasmid of pET28a-TsAab2 with C (Ara promoter). We synthesize two DNA primers which sequences are pairing with 5’ of based on sequencing results and gel picture comparing with marker, we can prove that the plasmid is successfully constructed, and plasmid size is 7943bp. The plasmid was successfully built and can be used in further construction of pET28a-TsAab2-UcFadD.

After enzyme digestion (NheI, XhoI and XbaI, XhoI) and ligation, we have successfully transfered the pET28a-TsAab2-UcFadD into E.coli. And the bacteria has formed single colony on Kanamycin LB medium, however we haven’t do the sequencing to verify the plasmid yet due to the limited time.

Step2

In this part, we designed and constructed a plasmid containing FadL gene and P450 BM3 gene sequences. After the plasmid was introduced into the FadD gene-deficient E. coli cells in the laboratory, the cells achieved increased absorption of exogenous long-chain fatty acids (LCFA). Then, hydroxylation reaction was carried out to make the host cells produce more hydroxyl fatty acids.

Fig1. Expected results of the experiment

Our objective was to hydroxylate LCFA which had already entered into the membrane. Thus, the presence of the FadD gene would hinder the experiment. By consulting the relevant literature, we found that Jin H and his team have done two experiments to prove that knockout of FadD gene and overexpression of Fadl in Escherichia coli can increase the production of hydroxylate in Escherichia coli. First, they knocked out the FadD gene in E. coli and introduced the P450 enzyme gene into the bacteria to convert palmitic acid into omega-hydroxypalmitic acid. The results of the experiments demonstrate that the production of ω-hydroxypalmitic acid is increased in the FadD-deleted, P450 overexpressed mutants. The experimental results are shown in fig. 3 (a). [1]

Later, they overexpressed Fadl gene on the basis of FadD gene deletion and P450 expression, and found that the production of ω-hydroxypalmitic acid was greatly increased. The experimental results are shown in fig. 3 (B). Because LCFA accumulates in the periplasm through FadL membrane protein transport and is consumed in the cytoplasm through P450 enzyme, the diffusion of LCFA is driven and will not be participated in β-Oxidation. As a result, the yield of hydroxylation products increase. [1]

Fig3. Experimental results by Jin H and his team[1]

After the completion of the plasmid design, we contacted the company to synthesize the fadL gene at the MCS I site of pCOLADuet-1, and linked it to the P450 gene by homologous recombination. At the same time, we extracted the plasmids which were successfully transferred into the cells and verified the vector part and P450 part by PCR. As shown in fig. 5.

Fig.5 The PCR result.
The first bright band is DNA Marker.
The second bright band is the vector part which has 4862 bp.
The third bright band is P450 which has 3150 bp.

Step3

We mainly want to express two proteins, CoA ligase and acyltransferase. The function of the two proteins as follows (EC for CoA ligase; WS2 for acyltransferase) At the same time, in our design, it contained a T7 promoter of EC between the two segments (EC / WS2)

In order to verify the functions of EC and WS2, we constructed the plasmid pETDuet (WS2/EC). In the verification experiment, we used hydroxydodecanoic acid and nonanoic acid as substrates. We expressed pETDuet (WS2/EC) into E. coli BL21 (DE3). The functions of EC and WS2 are shown below. During the polymerization process, EC acts as a CoA ligase to remove H in CoA-SH and -OH in the carboxyl group of hydroxy fatty acid through dehydration and condensation, and connect CoA and hydroxy fatty acid at the same time; WS2 As an acyltransferase, the acyl group in the hydroxy fatty acid CoA replaces the H in hydroxyl group of the hydroxy fatty acid, and CoA-SH is generated at the same time.

Experimental results and analysis

1.SDS protein electrophoresis

Molecular weight of target protein
EC 62 kDa
WS2 52.5 kDa
ACS2 61.9 kDa
Through SDS protein electrophoresis, we can confirm that our genes EC and WS2 are expressed, and the E.coli has the enzymes we need by comparing with marker. Which allowe us to conduct further experiments.

2.Results of GC-MS with Hydroxydodecanoic acid and nonanoic acid as substrates

After the engineering bacteria were successfully constructed, we added an exogenous substrate and added 3~4% DMSO to increase the solubility of the substrate hydroxy fatty acid and the permeability of the cell membrane. After the induction of fermentation with the IPTG inducer, the cells were broken and used Trichloromethane extracts the fermentation broth and prepares samples for gas phase detection. Set 1 group of control substrates nonanoic acid and hydroxydodecanoic acid (brown line) and 3 groups of experimental group substrates are 1. hydroxydodecanoic acid and isopropanol (blue line) 2. Nonanoic acid and hydroxydodecanoic acid (Purple line) 3. Nonanoic acid and isopropanol (black line).

According to the GC-MS spectrum, in addition to experimental group 3, the other two groups of experimental group 1 hydroxylauric acid and isopropanol (blue line) and experimental group 2 nonanoic acid and hydroxylauric acid (purple line) pass and control Dimeric hydroxy dodecanoic acid was found in the comparison of group nonanoic acid and hydroxydodecanoic acid (brown line), indicating that the polymerization pathway of engineered bacteria is feasible to polymerize hydroxydodecanoic acid; in addition, experimental group 2 nonanoic acid and hydroxydodecanoic acid ( Purple line) The product Nonanoic acid hydroxydodecanoate appears, which proves that the hydroxyl group of the hydroxy fatty acid can be used as the catalytic site of WS2 acyltransferase, and the carboxyl group at the other end does not affect the catalytic reaction of the acyltransferase. Our research has proved that the polymerization process of EC coenzyme A ligase and WS2 acyltransferase can play a role in two identical or different monomers and form at least two degree of polymerization. Due to the limitation of the experimental time and the experimental equipment caused by the epidemic, we have not been able to provide more evidence of terpolymers and above polymers. The vaporization temperature of the gas-phase analyzer we use is 380℃, which is such a temperature. It may not be possible to vaporize the higher poly hydroxy fatty acids and enter the analytical instrument, so we did not get the relevant data. In the next experiment, we plan to further increase the intracellular substrate concentration during fermentation and use a gas phase instrument with a higher vaporization temperature for product detection.

Step4

In this step, we want to synthesis functional collagen. The first gene sequence is the gene encoding prolyl hydroxylase enzyme from aquatic giant viruses. The second gene sequence is a fragment of human collagen type III COL3A1cDNA(hCOL3).We planned to use bacterially active prolyl hydroxylase enzymes from the giant virus mimivirus to modify collagen encoded by hCOL3 to produce hydroxylated collagen, which can provide a better cell culture

1.pET16b-L593 and pET28a-hCOL3 > plasmids

We found that some aquatic giant viruses, belonging to Phycodnaviridae and Mimiviridae, harbor genes encoding prolyl hydroxylase enzyme[2]. These viral hydroxylases are soluble and active when expressed in E. coli, thus opening new possibilities for the production of hydroxylated collagen in bacterial expression systems[3].We planed to use bacterially active prolyl hydroxylase enzymes from the giant virus mimivirus to produce hydroxylated collagen. For figure, it is the pET16b-L593 vector that we synthesized uses pET-16b as the expression vector and lacks His-Tag.

Primers and restriction Enzyme cutting sites:
5’-CCATGGATGAAAACTGTAACAATAATCACG-3’
5’-CGAACGTAAGTTCAGCTAAGGATCC-3’

Nucleic acid sequence

Tips:
(1) The red part is T7 promoter:
(2) The green part is Prolyl 4-hydroxylase [Acanthamoeba polyphaga mimivirus] gene (NCBI Reference Sequence: YP_003987108.1）
(3) The blue part is T7 terminator.

We chose to use a fragment of human collagen type III COL3A1cDNA, encompassing 1206 bp and lacking propeptide-encoding regions, was custom synthesized using codons optimized for bacterial expression and including NcoI and BamHI sites at 5’- and 3’-ends. The pET28a expression vector was first digested with NcoI-BamHI, which eliminates the His-tag at the 5N-terminal site. The resulting hCOL3 segment was inserted as a NcoI-BamHI fragment into pET28a, yielding pET28a-hCOL3 with His-tag.

Primers and restriction Enzyme cutting sites:
NcoI 5’-CCATGGATGTACGACAGTTATG-3’
BamHI 5’-CCACCATCACCACTAAGGATCC-3’

Nucleic acid sequence:

Tips:
(1) The red part is T7 promoter:
(2) The blue part is synthetic construct for human collagen gene, type III, alpha 1 (COL3A1 gene) fragment(hCOL3)(GenBank: HG779440.1);
(3) The yellow part is 6xHis tag gene sequence;
(4) The purple part is T7 terminator.

Protein sequence:

Tips:
(1) The green part is synthetic construct for human collagen, type III.
(2) The red part is 6xHis tag.

2. Experimental results and analysis
(1)SDS protein electrophoresis of expression of L593

We put pET16b-L593 into E. coli (BL21) and induced expression by adding IPTG at 18 °C and 30 °C.Through SDS protein electrophoresis, and comparing with marker, we can confirm the expression of L593.

2.SDS protein electrophoresis of purified hydroxylated collagen and content determination of hydroxylated collagen

The expressed hCOL3 protein and L593 enzyme were carried out enzyme modification reaction a suitable and vitro environment to obtain hydroxylated collagen. Because it has His-tag, it can be purified. The following picture shows SDS protein electrophoresis of purified hydroxylated collagen and content determination of hydroxylated collagen.

(Temperature-The times of elution product)

Through SDS protein electrophoresis, and comparing with marker, we can confirm the expression and purification of hydroxylated collagen.

Reference

[1] Bae Jin H; Park Beom Gi; Jung Eunok; Lee Pyung-Gang; Kim Byung-Gee. fadD deletion and fadL overexpression in Escherichia coli increase hydroxy long-chain fatty acid productivity[J].Applied Microbiology and BiotechnologyVolume 98, Issue 21. 2014. PP 8917-25
[2]Eriksson M, Myllyharju J, Tu H, Hellman M, Kivirikko KI (1999) Evidence for 4-
hydroxyproline in viral proteins. Characterization of a viral prolyl 4-hydroxylase and its
peptide substrates. J Biol Chem 274(32):22131-4
[3]Luther KB, Hulsmeier AJ, Schegg B, Deuber SA, Raoult D, Hennet T (2011) Mimivirus
collagen is modified by bifunctional lysyl hydroxylase and glycosyltransferase enzyme. J
Biol Chem 286(51):43701-9