Overview
Our project introduces an innovative approach to mosquito control by integrating two key elements: a high-impact mosquito attractant (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphat (HMBPP) and RNA interference (RNAi)-based mosquitocides within attractive targeted sugar baits (ATSBs). HMBPP is synthesized by the malaria parasite Plasmodium falciparum when infected with humans, acting as a potent attractant for blood-seeking mosquitoes. By integrating HMBPP with sugar baits, we aim to significantly increase their appeal to these mosquitoes, ensuring that they consume the bait. Once ingested, the designed shRNAs within the bait trigger the RNAi mechanism, leading to the mosquitoes' deaths.
Besides, in developing Moskilla, we proactively addressed potential biosafety concerns regarding live E. coli by incorporating autolysis genes. Through testing, we have identified the most effective candidate for this purpose.
To facilitate the selection of functional components by iGEM teams and researchers, we have comprehensively listed three distinct collections, ensuring a streamlined process for accessing the necessary elements for their projects.
HMBPP overproduction
In our efforts to enhance the production of HMBPP this year, we have successfully engineered variants of sRNA(IspH)s (BBa_K5186001-BBa_K5186005) for the downregulation of downstream gene IspH expression, and various MEP overexpression cassettes (BBa_K5186006-BBa_K5186009) utilising the main rate-limiting enzymes DXS(BBa_K3166061), DXR(BBa_K1950003), IspG(BBa_K1088004), IspDF(BBa_K1653001).
In the end,we have demonstrated about a 2.03-fold increase in the overexpression efficiency of PTac-riboJ-DXS-IspG-IspDF-B0015 (BBa_K5186008) with the help of the lycopene reporter, which makes it the best candidate for the MEP overexpression cassettes in our part collection. And "PTac-riboJ-DXS-IspG-IspDF-B0015-PTac-sRNA2(IspH)-B0015" (BBa_K5186005) reaches the highest yield of 0.45 mM of HMBPP without affecting bacterial growth in E. coli. This collection can help and inspire other iGEM teams and researchers to achieve higher yield of HMBPP or other isoprenoids in E. coli.
| Part name | Part number | Type | Description | Does It Function? | Group Favorite |
|---|---|---|---|---|---|
| sRNA 1(IspH) | BBa_K5186001 | RNA (New Basic Part) | The synthetic small RNAs(sRNAs) designed to down-regulate IspH in E. coli. | No | - |
| sRNA 2(IspH) | BBa_K5186002 | RNA (New Basic Part) | Yes | Yes | |
| sRNA 3(IspH) | BBa_K5186003 | RNA (New Basic Part) | No | - | |
| sRNA 4(IspH) | BBa_K5186004 | RNA (New Basic Part) | No | - | |
| PTac-riboJ-DXS-IspG-IspDF-B0015-PTac-sRNA2(IspH)-B0015 | BBa_K5186005 | New Composite Part | IPTG-inducible overexpression of DXS, IspG, IspDF and expression cassette of sRNA2(IspH) targeting IspH of E. coli for down-regulation. | Yes | Yes |
| PTac-riboJ-DXS-IspG-B0015 | BBa_K5186006 | New Composite Part | IPTG-inducible MEP overexpression cassettes for the higher yield of HMBPP. | Yes | - |
| PTac-riboJ-DXS-IspG-DXR-B0015 | BBa_K5186007 | New Composite Part | Yes | - | |
| PTac-riboJ-DXS-IspG-IspDF-B0015 | BBa_K5186008 | New Composite Part | Yes | Yes | |
| PTac-riboJ-DXS-IspG-DXR-IspDF-B0015 | BBa_K5186009 | New Composite Part | Yes | - | |
| PTac-riboJ-DXS-IspG-IspDF-B0015-PTac-sRNA1(IspH)-B0015 | BBa_K5186022 | New composite part | IPTG-inducible overexpression of DXS, IspG, IspDF and expression cassette of sRNA1, 3, 4(IspH) targeting IspH of E. coli for down-regulation. | No | - |
| PTac-riboJ-DXS-IspG-IspDF-B0015-PTac-sRNA3(IspH)-B0015 | BBa_K5186023 | New composite part | No | - | |
| PTac-riboJ-DXS-IspG-IspDF-B0015-PTac-sRNA4(IspH)-B0015 | BBa_K5186024 | New composite part | No | - | |
| DXS | BBa_K3166061 | Coding Sequence | A gene encoding 1-deoxy-D-xylulose 5-phosphate(DXP) synthase. | Yes | Yes |
| DXR | BBa_K1950003 | Coding Sequence (New Basic part) | A gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase. | Yes | - |
| IspG | BBa_K1088004 | Coding Sequence | A gene encoding HMBPP synthase. | Yes | Yes |
| IspDF | BBa_K1653001 | Coding Sequence | An operon made of IspD and IspF, encoding CDP-ME cytidylyltransferase and MEC synthase respectively. | Yes | Yes |
Targeted shRNAs for Efficient Mosquito Control
To combat mosquito populations, we utilized RNAi technique and engineered variants of short hairpin RNAs(shRNAs) to target mosquitoes' vital survival genes, including 5-HTR1(AAEL000528), Rbfox1 (AAEL019934), Shaker (AAEL000242) and Irx which encode serotonin(5-HT) receptors, evolutionarily conserved RNA binding proteins, an evolutionarily conserved subunit of voltage-gated potassium channels, and Iroquois-class homeodomain-containing proteins respectively.
This year, 6 shRNAs were demonstrated and thus make up a part collection for their contribution to mosquitoes control solutions. When these shRNAs in the freeze dried inactivated yeast cells are digested by mosquitoes, they will be processed into small interfering RNAs (siRNAs), and further specifically target the vital survival gene to achieve degradation and thereby causing mosquitoes' death without any harm to other non-target organisms.
This collection serves as a valuable resource for the iGEM community and researchers, offering a safe, efficient, and environmentally friendly approach to mosquito control.
| Part name | Part number | Type | Description | Does It Function? | Group Favorite |
|---|---|---|---|---|---|
| shRNA1(5-HTR1) | BBa_K5186011 | RNA (New Basic Part) | The shRNAs silencing 5-HTR1 gene of mosquitoes. | Yes | Yes |
| shRNA2(5-HTR1) | BBa_K5186012 | RNA (New Basic Part) | Yes | Yes | |
| shRNA3(5-HTR1) | BBa_K5186013 | RNA (New Basic Part) | Yes | Yes | |
| shRNA(Rbfox1) | BBa_K5186014 | RNA (New Basic Part) | The shRNAs silencing Rbfox1 gene of mosquitoes. | Yes | Yes |
| shRNA(shaker) | BBa_K5186015 | RNA (New Basic Part) | The shRNAs silencing Shaker gene of mosquitoes | Yes | Yes |
| shRNA(Irx) | BBa_K5186016 | RNA (New Basic Part) | The shRNAs silencing Irx gene of mosquitoes | Yes | Yes |
| pTDH3-shRNA(shaker)-tTDH1 | BBa_K5186017 | New Composite Part | IPTG-inducible expression cassette of shRNA(shaker). | Yes | Yes |
Autolytic Kill switch in E. coli
To address our potential end-users concerns towards biosafety of using live E. coli, we demonstrated several autolysis gene in E. coli for inducible kill switch setting, and thus make up this part collection.
To find out the most efficient autolysis gene, T4L(BBa_K5186018), Pa-T4L(BBa_K5186019), 2Pa-T4L(BBa_K5186020), X174E(BBa_K1835500) are engineered to be IPTG-inducibly expressed and tested. Our tests show that gene E outperforms T4L, Pa-T4L, and 2Pa-T4L, with the majority of cell lysis within one hour of induction. This makes gene E an optimal candidate for our kill switch component in the parts collection.
This collection serves as a valuable resource for the iGEM community and researchers for kill switch settings in E. coli and recovery of recombinant proteins or other intracellular products without the need for mechanical or chemical cell disruption methods.
| Part name | Part number | Type | Description | Does It Function? | Group Favorite |
|---|---|---|---|---|---|
| X174E | BBa_K1835500 | Coding Sequence | A gene encoding transmembrane pore-forming protein. | Yes | Yes |
| T4L | BBa_K5186018 | Coding Sequence (New Basic part) | A gene encoding T4 phage lysozyme. | Yes | - |
| Pa-T4L | BBa_K5186019 | Coding Sequence (New Basic part) | A gene encoding a fusion protein of an amphipathic Pa peptide and T4 phage lysozyme. | Yes | - |
| 2Pa-T4L | BBa_K5186020 | Coding Sequence (New Basic part) | A gene encoding a fusion protein of two amphipathic Pa peptides and T4 phage lysozyme. | Yes | - |
| Ptac-RiboJ-RBS-X174E-B0015 | BBa_K5186021 | New composite part | IPTG-inducible expression cassette of X174E. | Yes | Yes |