Team:HZAU-China/Engineering

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This year, we have summited five new parts to the registry to prove our Engineering Success.

ScGS

Gesomin synthase from Streptomyces coelicolor A3(2) (ScGS) is a single 726-amino acid protein which catalyzes the Mg²⁺ dependent conversion of farnesyl diphosphate to a mixture including geosmin. ScGS is a bifunctional enzyme in which the N-terminal domain catalyze the cyclization of FPP to form germacradienol, while the C-terminal domain then convert this sesquiterpenoid product to geosmin [1]. As it shows in the following figure (Figure 1), the existence of ScGS in our chasis was clearly proved by SDS-PAGE analysis.

Figure 1. SDS-PAGE analysis of ScGS with His-tag expression.

For further demonstration, we prepared the sample via headspace liguid-phase microextraction (HS-LPME) and a gas chromatography-mass spectrometry (GC-MS) test was conducted. The results given by GC-MS fairly shows the existence of geosmin in our culture (Figure 2), thus prove the feasibility of the part.

Figure 2.Identification of geosmin by GC-MS. A. Total ion current chromatogram of geosmin standard(Red Line) and extracted product(Blue line). B. Mass spectrum of geosmin standard. C. Mass spectrum of the extracted product.

For more details, please click the link below: http://parts.igem.org/Part:BBa_K3733006

LTA

LTA is a novel antimicrobial peptide (AMP), whose design was based on combing the active centers of a ride range of AMPs, including LL-37, YW12D, innate defense regulator 1, and cathelicidin 2 with thymopentin or the active center of thymosin alpha 1 (Tα1). It could neutralize Lipopolysaccharides (LPS), thus effectively blocking the downstream inflammation pathway. As it shows in the following figure (Figure 3), the existence of LTA in our chasis was clearly proved by Tricine-SDS-PAGE analysis [2].

Figure 3.Tricine-SDS-PAGE analysis of LTA.

We also confirmed the lytic activity of LTA by choosing Salmonella Typhimurium SL1344 as our test strain. The result is shown below (Figure 4)

Figure 4.OD₆₀₀-Time curve of Salmonella Typhimurium SL1344 in the presence or absence of LTA.

For more details, please click the link below: http://parts.igem.org/Part:BBa_K3733007

HyLα

HyLα is hybrid peptide of LL-37 and LTA. LL-37 and LTA are both affective antimicrobial peptides (AMPs). successfully combine those two AMPs by connecting the C-terminate of LTA with the N-terminate of LL-37 through GS-linker. It could neutralize Lipopolysaccharides (LPS), thus effectively blocking the downstream inflammation pathway. As it shows in the figure (Figure 3), the existence of HyLα in our chasis was clearly proved by Tricine-SDS-PAGE analysis. We also confirmed the lytic activity of HyLα by choosing Salmonella Typhimurium SL1344 as our test strain. The result is shown below (Figure 5)

Figure 5.OD₆₀₀-Time curve of Salmonella Typhimurium SL1344 in the presence or absence of HyLα.

For more details, please click the link below: http://parts.igem.org/Part:BBa_K3733008

HepT

HepT is a toxin, a member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) superfamily, strongly inhibited cell growth in S.oneidensis and Escherichia coli. The HepT toxin (HEPN-domain protein) could function as an RNase with a RX4-6H active motif and cleave mRNA to inhibit cell growth [3]. The cytotoxicity of HepT is shown in the figure (Figure 6).

Figure 6. A. Turbidity change of the experimental group (induced by IPTG) and control group (without IPTG). B. The result of OD600 (-IPTG and +IPTG) presents the cytotoxicity of HepT. The error bars are obtained from three independent experiments.

For more details, please click the link below: http://parts.igem.org/Part:BBa_K3733010

RNA Thermometer_NoHeat

This basic part is one of heat-repressible RNA thermosensor which could inhibit downstream gene expression when the temperature is 37 ℃ but not affect downstream gene expression significantly when the temperature is below 28 ℃. The function of this RNA thermometer is based on the stem structure which was created by taking the complement of the RNase-binding sequence. At low temperatures, the RNase-binding sequence is protected by the stem structure and downstream gene could be expressed normally. At high temperatures, the stem is destabilized, which allows RNase E (endogenic in E. coli) to bind with the RNase-binding sequence and turn off downstream gene expression [4]. We verified the function of this part in our project, the result is shown below (Figure 7).

Figure 7. A. The comparison photo of the experimental group (toxin system) and control group incubated at both 37 ℃ and 28 ℃ for 12 hours. NoHeat means the experimental group (toxin system). Control means the control group. B. The specific OD600 data of the experimental group and control group.

For more details, please click the link below: http://parts.igem.org/Part:BBa_K3733011

Reference:

[1] Harris G G, Lombardi P M, Pemberton T A, et al. Structural Studies of Geosmin Synthase, a Bifunctional Sesquiterpene Synthase with αα Domain Architecture That Catalyzes a Unique Cyclization–Fragmentation Reaction Sequence[J]. Biochemistry, 2015, 54(48): 7142-7155.

[2]Zhang L, Wei X, Zhang R, et al. Design and development of a novel peptide for

treating intestinal inflammation [J]. Frontiers in immunology, 2019, 10: 1841.

[3] Yao J, Zhen X, Tang K, et al. Novel polyadenylylation-dependent neutralization mechanism of the HEPN/MNT toxin/antitoxin system[J]. Nucleic acids research, 2020, 48(19): 11054-11067.

[4] Hoynes-O'Connor A, Hinman K, Kirchner L, et al. De novo design of heat-repressible RNA thermosensors in E. coli[J]. Nucleic acids research, 2015, 43(12): 6166-6179.