Difference between revisions of "Part:BBa K5186004"

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sRNA4(IspH)

Description

sRNA(IspH)s are synthetic small regulatory RNAs (sRNAs) designed to specifically down-regulate the expression of the IspH gene in E. coli, thereby enhancing HMBPP production for mosquito attraction. Each sRNA(IspH) is composed of a target-binding sequence that is complementary to the IspH mRNA, enabling hybridization, and a SgrS scaffold that facilitates the recruitment of the Hfq protein in E. coli. This interaction promotes the sRNAs' binding to IspH mRNA and expedites its degradation by RNase E. (Seung Min Yoo et al., 2013)

It has been revealed that the mutation in the SgrS scaffold can enhance the efficiency of sRNA-mediated gene repression (Minho Noh et al., 2019). This year, we have successfully engineered 4 variants of sRNA(IspH)s in E. coli, each with a specialized scaffold. We expect to select the best one for controlling the down-regulation of IspH from them. sRNA4(IspH) incorporates a CUUU-6nt stem with an additional GUCU sequence in the SgrS-S scaffold, while sRNA2(IspH) with a SgrS-S 6-nts stem scaffold exhibits the most excellent downregulation effect without affecting bacterial growth (BBa_K5186002).

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.). 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 design of sRNAs is mainly inspired by trans-acting Hfq-dependent sRNAs, which binds at or near the ribosome-binding sites(RBSs) of target mRNAs with the aid of the RNA charperone Hfq. This binding prevents ribosomes from accessing the RBS, thereby inhibiting translation. (Seung Min Yoo et al., 2013)

In our study, sRNA4(IspH) is constitutively expressed under the control of pTac while overexpressing DXS, IspG and IspDF. Upon expression, sRNA4(IspH) binds to Hfq protein, aligning its target-binding sequence with the IspH mRNA. This interaction prevents ribosomes access to the RBS, effectively downregulating IspH expression and allowing for precise control over HMBPP synthesis without decreasing E. coli's viability.

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.

To identify the most effective candidate for IspH downregulation, we designed 4 variants of sRNA(IspH)s, each with a specialized scaffold derived from SgrS, as is shown in Figure 1b. PCR and gel electrophoresis verified the successful construction of these sRNA(IspH)s overexpression cassettes (Figure 1c), and growth curves demonstrated that the designed sRNA(IspH) expression did not impede bacterial growth (Figure 1d), 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.

To determine the most effective sRNA candidate for IspH downregulation, the four sRNA(IspH) variants were co-expressed with the MEP overexpression cassette 3, comprising DXS, IspG and IspDF, and HMBPP yields 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, E. coli strain DH5a, indicating that MEP overexpression significantly enhanced HMBPP production (Figure 2).

Furthermore, HMBPP levels in all samples are quantified based on LC-MS analysis. Contrary to our expectations, the presence of sRNA1(IspH), sRNA3(IspH), and sRNA4(IspH) resulted in HMBPP yields that were 17.46%, 23.03%, and 13.80% lower, respectively, than the positive control. However, sRNA2(IspH), featuring a SgrS-S 6-nts stem scaffold, achieved the most notable downregulation effect, increasing HMBPP yield by approximately 1.1-fold.

The superior performance of sRNA2(IspH) confirms our integrated approach, combining MEP pathway overexpression with sRNA-mediated IspH 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.
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.

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
COMPATIBLE WITH RFC[21]
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COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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-   <p style="font-size: smaller; margin-top: 10px;"> Figure 2. 4 variants of sRNA(IspH)s are engineered in <i>E. coli</i> for down-regulation of <i>IspH</i>. (a) A graphical abstract of the molecular mechanism underlying the down-regulation of <i>IspH</i> expression by <i>sRNA</i>(IspH)s. (b) Genetic circuit and nucleotide sequences of <i>sRNA</i>(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 <i>sRNA</i>(IspH)s expression cassettes. (d) Growth curve of control (<i>E. coli</i> strain DH5a) and strains expressing <i>sRNA</i>(IspH)s.</p>
+   <p style="font-size: smaller; margin-top: 10px;"> Figure 2. 4 variants of sRNA(IspH)s are engineered in <i>E. coli</i> for down-regulation of <i>IspH</i>. (a) A graphical abstract of the molecular mechanism underlying the down-regulation of <i>IspH</i> 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 (<i>E. coli</i> strain DH5a) and strains expressing sRNA(IspH)s.</p>
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-Furthermore, HMBPP levels in all samples are quantified based on LC-MS analysis. Contrary to our expectations, the presence of <i>sRNA1</i>(IspH), <i>sRNA3</i>(IspH), and <i>sRNA4</i>(IspH) resulted in HMBPP yields that were 17.46%, 23.03%, and 13.80% lower, respectively, than the positive control. However, <i>sRNA2</i>(IspH), featuring a SgrS-S 6-nts stem scaffold, achieved the most notable downregulation effect, increasing HMBPP yield by approximately 1.1-fold.
+Furthermore, HMBPP levels in all samples are quantified based on LC-MS analysis. Contrary to our expectations, the presence of sRNA1(IspH), sRNA3(IspH), and sRNA4(IspH) resulted in HMBPP yields that were 17.46%, 23.03%, and 13.80% lower, respectively, than the positive control. However, sRNA2(IspH), featuring a SgrS-S 6-nts stem scaffold, achieved the most notable downregulation effect, increasing HMBPP yield by approximately 1.1-fold.
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    <p style="font-size: smaller; margin-top: 10px;"> Figure 2. LC-MS analysis of the fermentation product of Negative control (NC), Positive control (PC) and sRNA1-4.
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-Notes: 1. NC indicates <i>E. coli</i> strain DH5a as negative control. 2. PC indicates <i>E. coli</i> strain DH5a with <i>DXS, IspD, IspF, IspG</i> overexpression cassette as positive control. 3. sRNA1-4 indicate <i>E. coli</i> strain DH5a with <i>sRNA1</i>(IspH), <i>sRNA2</i>(IspH), <i>sRNA3</i> (IspH), <i>sRNA4</i> (IspH) respectively while overexpressing <i>DXS, IspD, IspF, IspG</i>.</p>
+Notes: 1. NC indicates <i>E. coli</i> strain DH5a as negative control. 2. PC indicates <i>E. coli</i> strain DH5a with <i>DXS, IspD, IspF, IspG</i> overexpression cassette as positive control. 3. sRNA1-4 indicate <i>E. coli</i> strain DH5a with sRNA1(IspH), sRNA2(IspH), sRNA3(IspH), sRNA4(IspH) respectively while overexpressing <i>DXS, IspD, IspF, IspG</i>.</p>
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