Part:BBa_K5186011

shRNA1(5-HTR1)

Description

To combat mosquito populations, shRNAs (5-HTR1), variants of short hairpin RNAs, are engineered to target mosquitoes' 5-HTR1 gene, which encodes serotonin(5-HT) receptors crucial for immune and nervous systems, utilizing RNAi technique. When shRNA1(5-HTR1) in the freeze dried inactivated yeast cells are digested by mosquitoes, they will be processed into small interfering RNAs (siRNAs), and further specifically target mRNA(5-HTR1) starting at 1666 nt to achieve degradation. This targeted interference results in neural misfunctions in mosquitoes, eventually causing their deaths without any harm to other non-target organisms.

These shRNAs(5-HTR1) (BBa_K5186011, BBa_K5186012, BBa_K51860013) and other shRNAs targeting mosquitoes' vital survival genes (BBa_K5186014, BBa_K5186015, BBa_K5186016, BBa_K5186017) contribute to mosquitoes control solutions and thus make up a part collection. This collection serves as a valuable resource for the iGEM community and researchers, offering a safe, efficient, and environmentally friendly approach to mosquito control.

Biology

The gene 5-HTR1(AAEL000528) encodes serotonin(5-HT) receptors, which are located on the cell membrane of neurons and other cell types. In insects, 5-HT receptors play a pivotal role in regulating sleep, feeding, the circadian rhythm, and cognitive functions such as learning and memory, thereby influencing a range of insect behaviors and physiological processes. It has been proved that the silence of the 5-HTR1 gene can lead to significant neural defects in the brains of mosquitoes.

Design

We utilized a professional shRNA designed website(https://rnaidesigner.thermofisher.com/rnaiexpress/) to craft shRNAs targeting the 5-HTR1 gene, which is composed of sense strand, loop and antisense strand.

To address biosafety concerns, aftering gaining the shRNAs candidates, we conducted BLAST searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) of the sense strand sequences against the total RNA database, ensuring their specificity to mosquitoes and no target to other organisms.

Finally, we arranged for the synthesis of DNA oligos encoding the shRNAs(5-HTR1), which were then assembled into the multiple cloning site of the pRS426 yeast shuttle vector, located downstream of pTDH3 and upstream of tTDH1.

Table 1. List of shRNAs(5-HTR1)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]