Composite

Part:BBa_K5186024

Designed by: Jiawen Chen   Group: iGEM24_AIS-China   (2024-09-30)
Revision as of 03:47, 3 December 2024 by Garmen (Talk | contribs)


PTac-riboJ-DXS-IspG-IspDF-B0015-PTac-sRNA4(IspH)-B0015

Description

“PTac-riboJ-DXS-IspG-IspDF-B0015-PTac-sRNA4(IspH)-B0015” is an expression cassette in E. coli expressing sRNA4(IspH) and overexpress DXS, IspG, IspDF. With this expression cassette, sRNA4(IspH), DXS, IspG and IspDF can be IPTG-inducibly expressed under the control of pTac and in all strains of E. coli. It is designed to enhance HMBPP production for mosquito attraction. However, the expression cassette of sRNA2(IspH) (BBa_K5186005) leads to the highest yield of HMBPP without affecting bacterial growth in E. coli.

This is a part of a part collection where we enable the overproduction of HMBPP. The part collection includes the sRNA(IspH)s variants and their expression cassettes (BBa_K5186001, BBa_K5186002, BBa_K5186003, BBa_K5186004, BBa_K5186005, BBa_K5186022, BBa_K5186023, BBa_K5186024) for the downregulation of downstream gene IspH expression, and various MEP overexpression cassettes (BBa_K5186006, BBa_K5186007, BBa_K5186008, BBa_K5186009). This collection can help and inspire other iGEM teams and researchers to achieve higher yield of HMBPP or other isoprenoids in E. coli.

Usage and biology

In E. coli, IspH is an essential enzyme in the MEP pathway, crucial to isoprenoid synthesis which helps maintain, stabilize and support core functions such as respiration. It catalyzes the conversion of HMBPP into the common isoprenoid precursors IPP and DMAPP in a single step. And it has been confirmed that significantly inhibiting the IspH activity in E. coli substantially resctricts the bacterial growth (Kumar et al., 2021). To achieve a balance between HMBPP overproduction and growth activity of E. coli, we decided on the sRNA-based down-regulation of IspH instead of the knockout approach.

The synthetic small RNA (sRNA) sRNA4(IspH) (BBa_K5186001), which targets the gene IspH<i>, was then designed to encode sRNA. Upon expression, sRNA4(IspH) with CUUU-6nts stem and GUCU mutation in SgrS-S scaffold binds to the Hfq protein, aligning its target-binding sequence with the <i>IspH<i> mRNA. This interaction prevents ribosomes from accessing the ribosome binding site (RBS), effectively downregulating <i>IspH<i> expression and allowing for precise control over HMBPP synthesis without compromising the viability of <i>E. coli.

In our efforts to increase HMBPP production this year, we successfully designed an expression cassette for sRNA4 (IspH) in E. coli DH5a, based on overexpression of the MEP pathway. Nevertheless, it leads to a reduction in HMBPP yields by 13.80%, which is contrary to our expectation.

Source

DXS (BBa_K3166061), IspG (BBa_K1653001) and IspDF (BBa_K1653001) are from E. coli. sRNA4(IspH) is synthetic.

Characterization

In our project, HMBPP is used to attract blood-feeding mosquitoes. Since HMBPP cannot be chemically synthesized, we selected E. coli as the chasis for HMBPP production, utilizing its inherent MEP pathway, which is similar to that of Plasmodium (Emami et al., 2017; Viktoria et al., 2021). To enhance HMBPP yield, we implemented dual metabolic engineering strategies: overexpression of the upstream genes in the MEP pathway and downregulating the expression of the downstream IspH enzyme.

Co-expressing with lycopene expression cassette as reporter, various MEP overexpression cassettes were demonstrated by measuring the A470/A600 ratio to analyze lycopene production per cell unit for the selection of the most promising candidate. All strains 1-4 demonstrated a notable increase in lycopene yield relative to the control strain with the reporter cassette alone. Notably, strain 3, harboring the MEP overexpression cassette 3, outperformed with a 2.03-fold enhancement in overexpression efficiency, indicating that the combination of DXS, IspG, and IspDF is the most promising candidate. (Figure 1d)

Figure 1. Using lycopene as reporter, the best MEP overexpression cassette is selected for higher yield of HMBPP. (a) Various MEP pathway overexpression cassettes expression in E. coli strain DH5a (b) Production of lycopene via the endogenous MEP pathway in E. coli. (c) Gel electrophoresis analysis of transformed MEP pathway overexpression cassettes. (d) Relative lycopene production while using various MEP Overexpression Cassettes in E. coli.The data are the means ± SD of three parallel replicate experiments. Statistical significance comparing conditions with and without MEP Overexpression Cassette use one-way ANOVA test. ***P< 0.0001.


To identify the most effective candidate for IspH<i> downregulation, we designed 4 variants of sRNA(IspH)s, each with a specialized scaffold derived from SgrS, as is shown in Figure 2b. PCR and gel electrophoresis verified the successful construction of these sRNA(IspH)s overexpression cassettes (Figure 2c), and growth curves demonstrated that the designed sRNA(IspH) expression did not impede bacterial growth (Figure 2d), validating the potential of our sRNA-mediated strategy.

Figure 2. 4 variants of sRNA(IspH)s are engineered in E. coli for down-regulation of IspH. (a) A graphical abstract of the molecular mechanism underlying the down-regulation of IspH expression by sRNA(IspH)s. (b) Genetic circuit and nucleotide sequences of sRNA(IspH)s expression. The green and blue sequences indicate the target-binding sequences and SgrS-S scaffold variants respectively. (c) Gel electrophoresis analysis of transformed sRNA(IspH)s expression cassettes. (d) Growth curve of control (E. coli strain DH5a) and strains expressing sRNA(IspH)s.The data are the means ± SD of three parallel replicate experiments.


To determine the most effective sRNA candidate for <i>IspH<i> downregulation, the four sRNA(IspH) variants expression cassettes were assembled to the MEP overexpression cassette 3, containing <i>DXS, IspG, and IspDF. The E. coli strain DH5a carrying these composite expression cassettes were then designated as sRNA1, sRNA2, sRNA3 and sRNA4, respectively. The yields of HMBPP were measured using Liquid Chromatography-Mass Spectrometry (LC-MS).

The LC-MS results for the HMBPP standard revealed a mass/charge ratio (m/z) of approximately 260.993. HMBPP was detectable in the majority of the fermentation products, with the exception of the negative control, indicating that MEP overexpression significantly enhanced HMBPP production (Figure 2).

Unexpectedly, the introduction of sRNA1(IspH), sRNA3(IspH), and sRNA4(IspH) led to reductions in HMBPP yields by 17.46%, 23.03%, and 13.80%, respectively, compared to the positive control.

However, the expression of sRNA2(IspH), with its SgrS-S 6-nts stem scaffold, achieved the most notable downregulation effect, increasing HMBPP yield by approximately 1.1-fold with the highest yield of 0.45 mM.

The superior performance of sRNA2 confirms our integrated approach, combining MEP pathway overexpression with sRNA-mediated IspH<i> downregulation, as an effective strategy to enhance HMBPP production (Figure 3).

Figure 2. LC-MS analysis of the fermentation product of Negative control (NC), Positive control (PC) and sRNA1-4. Notes: 1. NC indicates E. coli strain DH5a as negative control. 2. PC indicates E. coli strain DH5a with DXS, IspD, IspF, IspG overexpression cassette as positive control. 3. sRNA1-4 indicate E. coli strain DH5a with sRNA1(IspH), sRNA2(IspH), sRNA3(IspH), sRNA4(IspH) respectively while overexpressing DXS, IspD, IspF, IspG.



Figure 3. Analysis of HMBPP production when expressing various sRNA(IspH)s. (a) Standard curve of HMBPP. (b) The yield of HMBPP in NC, PC, sRNA1-4 fermentation products. The data are the means ± SD of three parallel replicate experiments. Statistical significance comparing conditions with and without sRNA Expression Cassette use one-way ANOVA test. ***P< 0.0001.
Note: The fact that the HMBPP concentration in the NC product is 0 does not imply the absence of HMBPP; rather, it suggests that its concentration is below the LC-MS instrument's detection threshold.


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Reference

Kumar S. S., Rishabh S., Poli Adi N. R., Prashanthi V., et al. IspH inhibitors kill Gram-negative bacteria and mobilize immune clearance. Nature. 2021, 589(7843): 597-602. https://www.nature.com/articles/s41586-020-03074-x

Seung Min Yoo, Dokyun Na, Sang Yup Lee. Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli. Nat Protoc. 2013, 8(9): 1694-707. https://www.nature.com/articles/nprot.2013.105

Minho Noh, Seung Min Yoo, Dongsoo Yang, Sang Yup Lee. Broad-Spectrum Gene Repression Using Scaffold Engineering of Synthetic sRNAs. ACS synth Biol. 2019, 8(6): 1452-1461. https://pubs.acs.org/doi/10.1021/acssynbio.9b00165

Emami, S. N., Lindberg, B. G., Hua, S., Hill, S. R., Mozuraitis, R., Lehmann, P., Birgersson, G., Borg-Karlson, A.-K., Ignell, R., & Faye, I. A key malaria metabolite modulates vector blood seeking, feeding, and susceptibility to infection. Sci. 2017, 355(6329): 1076-1080. https://doi.org/doi:10.1126/science.aah4563

Viktoria, E. S., Melika, H., Elizabeth, V., Raimondas, M. , S. Noushin, E. Plasmodium metabolite HMBPP stimulates feeding of main mosquito vectors on blood and artificial toxic sources. Commun. Biol. 2021, 4(1): 1161. https://www.nature.com/articles/s42003-021-02689-8

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4325
    Illegal BglII site found at 5541
    Illegal BamHI site found at 3653
    Illegal BamHI site found at 4869
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2102


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