shRNA_v1: BBa_K5047019

Short description
DNA encodes a shRNA that does not bind to any genes of V. velutina. It serves as a control.

Long description
This DNA sequence encodes a shRNA which can be used as a control in RNA interference experiments. Instead of targeting a functional gene this sequence contains two inverted repeats of a sgRNA derived from a CRISPR interference system separated by a 9nt loop Brummelkamp, Thijn R et al. “A system for stable expression of short interfering RNAs in mammalian cells.” Science (New York, N.Y.) vol. 296,5567 (2002): 550-3. doi:10.1126/science.1068999. The shRNA does not have any significant biological target in our model organism making it an ideal negative control to verify the specificity and functionality of the RNA interference RNAi machinery in our experiments. This ensures that any observed effects in the system are due to the specific shRNAs being tested and not due to nonspecific or off-target interactions.

Source
This part was synthetically designed. The two inverted repeats correspond to sgRNA_1 part of a CRISPR interference system published by Santos-Moreno, Javier et al. “Multistable and dynamic CRISPRi-based synthetic circuits.” Nature communications vol. 11,1 2746. 2 Jun. 2020, doi:10.1038/s41467-020-16574-1. The 9nt loop was published by Brummelkamp, Thijn R et al. “A system for stable expression of short interfering RNAs in mammalian cells.” Science (New York, N.Y.) vol. 296,5567 (2002): 550-3. doi:10.1126/science.1068999.

Design consideration The sequence was chosen because it lacks any significant homology to functional genes in our target organism, the Asian hornet, thereby avoiding off-target effects making it an appropriate negative control.

Promoter PTSIIC: BBa_K5047020

Short description
Constitutive promoter in Lactococcus lactis.

Long description
This promoter is an endogenous constitutive promoter from L. lactis, characterized by Ogaugwu et al. The author demonstrated that cellobiose enhances activity from PTS-IIC promoter and that it can mediate protein expression in B. subtilis and E. coli Nissle 1917.

In our project, we used the PTSIIC promoter to express shRNA in cells to trigger RNA interference.

Source
Part synthetically designed according to the sequence reported by Ogaugwu et al.

Design consideration
None.

shRNA TS1_Hairpin1: BBa_K5047030

Short description
DNA encodes a shRNA that binds to the chitin synthase gene of V. velutina.

Long description
This DNA sequence encodes a short hairpin RNA shRNA designed to target a specific region within the chitin synthase gene of the Asian hornet Vespa velutina. The target region is located on an exon common to all isoforms of the chitin synthase gene GeneID 124951980 Genbank XM_047501172.1 within the genomic coordinates NC_062188.1 19915311-19930067 iVesVel2.1 genome GCF_912470025.1. The specific target sequence spans from positions 19925853 to 19925878. This shRNA has been designed to keep GC content intermediate approx. 30 - 50% with 48% in this variant to avoid strong secondary structure. Additionally, this shRNA is the variant with the lowest number of potential off-target interactions compared to TS2 BBa_K5047021 TS3 BBa_K5047024 and TS4 BBa_K5047027. This sequence has off-targets in Vespa mandarinia and Vespa crabro with one mismatch only. This shRNA contains the standard UUCAAGAGA 9 nt loop Brummelkamp, Thijn R et al. “A system for stable expression of short interfering RNAs in mammalian cells.” Science (New York, N.Y.) vol. 296,5567 (2002): 550-3. doi:10.1126/science.1068999. Through secondary structure analysis performed by ViennaRNA the free energy of the thermodynamic ensemble is -48.96 kcal/mol. The frequency of the minimum free energy structure in the ensemble is 40.08%. The ensemble diversity is 1.71.

Source
This part was synthetically designed within the context of this project.

Design consideration
None.

shRNA TS1_Hairpin2: BBa_K5047031

Short description
DNA encodes a shRNA that binds to the chitin synthase gene of V. velutina.

Long description
This DNA sequence encodes a short hairpin RNA shRNA designed to target a specific region within the chitin synthase gene of the Asian hornet Vespa velutina. The target region is located on an exon common to all isoforms of the chitin synthase gene GeneID 124951980 Genbank XM_047501172.1 within the genomic coordinates NC_062188.1 19915311-19930067 iVesVel2.1 genome GCF_912470025.1. The specific target sequence spans from positions 19925853 to 19925878. This shRNA has been designed to keep GC content intermediate approx. 30 - 50% with 48% in this variant to avoid strong secondary structure. Additionally, this shRNA is the variant with the lowest number of potential off-target interactions compared to TS2 BBa_K5047022 TS3 BBa_K5047025 and TS4 BBa_K5047028. This sequence has off-targets in Vespa mandarinia and Vespa crabro with one mismatch only. Upon testing in human embryonic kidney 293T cells using a luciferase assay, Schopman Nick C T et al. “Optimization of shRNA inhibitors by variation of the terminal loop sequence.” Antiviral Research vol. 86.2 (2010): 204-11. doi: 10.1016/j.antiviral.2010.02.320 reported that shRNAs based on the mir-17 and mir-25 loops exhibited the strongest target repression, approximately 10-fold stronger than the 9-nt loop. This shRNA contains the optimized UGAUAUGUGCA loop Schopman Nick C T et al. Optimization of shRNA inhibitors by variation of the terminal loop sequence Antiviral research vol 86.2 2010 204-11 doi: 10.1016/j.antiviral.2010.02.320. Through secondary structure analysis performed by ViennaRNA the free energy of the thermodynamic ensemble is -50.39 kcal/mol. The frequency of the minimum free energy structure in the ensemble is 52.98%. The ensemble diversity is 0.70.

Source
This part was synthetically designed within the context of this project.

Design consideration
None.

shRNA TS1_Hairpin3: BBa_K5047032

Short description
DNA encodes a shRNA that binds to the chitin synthase gene of V. velutina.

Long description
This DNA sequence encodes a short hairpin RNA (shRNA) designed to target a specific region within the chitin synthase gene of the Asian hornet *Vespa velutina*. The target region is located on an exon common to all isoforms of the chitin synthase gene (GeneID: 124951980, Genbank XM_047501172.1) within the genomic coordinates NC_062188.1 19915311-19930067 of the iVesVel2.1 genome (GCF_912470025.1). The specific target sequence spans from positions 19925853 to 19925878. This shRNA has been designed to maintain an intermediate GC content of approximately 30 - 50%, with 48% in this variant, to avoid strong secondary structures. Additionally, this shRNA is the variant with the lowest number of potential off-target interactions compared to TS2 (BBa_K5047023), TS3 (BBa_K5047026), and TS4 (BBa_K5047029). This sequence has off-targets in *Vespa mandarinia* and *Vespa crabro* with only one mismatch. Upon testing in human embryonic kidney 293T cells using a luciferase assay, Schopman Nick C T et al. reported that shRNAs based on the mir-17 and mir-25 loops exhibited the strongest target repression, approximately 10-fold stronger than the 9-nt loop. This shRNA contains the optimized CCUCUCAACACUGG loop. Through secondary structure analysis performed by ViennaRNA, the free energy of the thermodynamic ensemble is -53.09 kcal/mol, with the frequency of the minimum free energy structure in the ensemble at 53.13%. The ensemble diversity is 0.69.

Source
This part was synthetically designed within the context of this project.

Design consideration
None.

shRNA TS2_Hairpin1: BBa_K5047021

Short description
DNA encodes a shRNA that binds to the chitin synthase gene of V. velutina.

Long description
This DNA sequence encodes a short hairpin RNA (shRNA) designed to target a specific region within the chitin synthase gene of the Asian hornet *Vespa velutina*. The target region is located on an exon common to all isoforms of the chitin synthase gene (GeneID: 124951980, Genbank XM_047501172.1) within the genomic coordinates NC_062188.1 19915311-19930067 of the iVesVel2.1 genome (GCF_912470025.1). The specific target sequence spans from positions 19925844 to 19925869. This shRNA has been designed to maintain an intermediate GC content of approximately 30 - 50%, with 44% in this variant, to avoid strong secondary structures. This sequence has off-targets in *Vespa mandarinia* (one mismatch), *Vespa crabro* (one INDEL), and *Rhorus exstirpatorius* (one mismatch). This shRNA contains the standard UUCAAGAGA 9-nt loop (Brummelkamp, Thijn R et al. “A system for stable expression of short interfering RNAs in mammalian cells.” *Science* (New York, N.Y.) vol. 296,5567 (2002): 550-3. doi:10.1126/science.1068999). Through secondary structure analysis performed by ViennaRNA, the free energy of the thermodynamic ensemble is -45.96 kcal/mol, with the frequency of the minimum free energy structure in the ensemble at 40.26%. The ensemble diversity is 1.69.

Source
This part was synthetically designed within the context of this project.

Design consideration
None.

shRNA TS2_Hairpin2: BBa_K5047022

Short description
DNA encodes a shRNA that binds to the chitin synthase gene of V. velutina.

Long description
This DNA sequence encodes a short hairpin RNA (shRNA) designed to target a specific region within the chitin synthase gene of the Asian hornet *Vespa velutina*. The target region is located on an exon common to all isoforms of the chitin synthase gene (GeneID: 124951980, Genbank XM_047501172.1) within the genomic coordinates NC_062188.1 19915311-19930067 of the iVesVel2.1 genome (GCF_912470025.1). The specific target sequence spans from positions 19925844 to 19925869. This shRNA has been designed to maintain an intermediate GC content of approximately 30 - 50%, with 44% in this variant, to avoid strong secondary structures. This sequence has off-targets in *Vespa mandarinia* (one mismatch), *Vespa crabro* (one INDEL), and *Rhorus exstirpatorius* (one mismatch). Upon testing in human embryonic kidney 293T cells using a luciferase assay, Schopman Nick C T et al. “Optimization of shRNA inhibitors by variation of the terminal loop sequence.” *Antiviral Research* vol. 86.2 (2010): 204-11. doi: 10.1016/j.antiviral.2010.02.320 reported that shRNAs based on the mir-17 and mir-25 loops exhibited the strongest target repression, approximately 10-fold stronger than the 9-nt loop. This shRNA contains the optimized UGAUAUGUGCA loop (Schopman Nick C T et al. *Optimization of shRNA inhibitors by variation of the terminal loop sequence* Antiviral research vol 86.2 2010 204-11 doi: 10.1016/j.antiviral.2010.02.320). Through secondary structure analysis performed by ViennaRNA, the free energy of the thermodynamic ensemble is -47.39 kcal/mol, with the frequency of the minimum free energy structure in the ensemble at 53.22%. The ensemble diversity is 0.68.

Source
This part was synthetically designed within the context of this project.

Design consideration
None.

shRNA TS2_Hairpin3: BBa_K5047023

Short description
DNA encodes a shRNA that binds to the chitin synthase gene of V. velutina.

Long description
This DNA sequence encodes a short hairpin RNA (shRNA) designed to target a specific region within the chitin synthase gene of the Asian hornet *Vespa velutina*. The target region is located on an exon common to all isoforms of the chitin synthase gene (GeneID: 124951980, Genbank XM_047501172.1) within the genomic coordinates NC_062188.1 19915311-19930067 of the iVesVel2.1 genome (GCF_912470025.1). The specific target sequence spans from positions 19925844 to 19925869. This shRNA has been designed to maintain an intermediate GC content of approximately 30 - 50%, with 44% in this variant, to avoid strong secondary structures. This sequence has off-targets in *Vespa mandarinia* (one mismatch), *Vespa crabro* (one INDEL), and *Rhorus exstirpatorius* (one mismatch). Upon testing in human embryonic kidney 293T cells using a luciferase assay, Schopman Nick C T et al. “Optimization of shRNA inhibitors by variation of the terminal loop sequence.” *Antiviral Research* vol. 86.2 (2010): 204-11. doi: 10.1016/j.antiviral.2010.02.320 reported that shRNAs based on the mir-17 and mir-25 loops exhibited the strongest target repression, approximately 10-fold stronger than the 9-nt loop. This shRNA contains the optimized CCUCUCAACACUGG loop (Schopman Nick C T et al. *Optimization of shRNA inhibitors by variation of the terminal loop sequence* Antiviral research vol 86.2 2010 204-11 doi: 10.1016/j.antiviral.2010.02.320). Through secondary structure analysis performed by ViennaRNA, the free energy of the thermodynamic ensemble is -50.09 kcal/mol, with the frequency of the minimum free energy structure in the ensemble at 53.36%. The ensemble diversity is 0.68.

Source
This part was synthetically designed within the context of this project.

Design consideration
None.

shRNA TS3_Hairpin1: BBa_K5047024

Short description
DNA encodes a shRNA that binds to the chitin synthase gene of V. velutina.

Long description
This DNA sequence encodes a short hairpin RNA (shRNA) designed to target a specific region within the chitin synthase gene of the Asian hornet *Vespa velutina*. The target region is located on an exon common to all isoforms of the chitin synthase gene (GeneID: 124951980, Genbank XM_047501172.1) within the genomic coordinates NC_062188.1 19915311-19930067 of the iVesVel2.1 genome (GCF_912470025.1). The specific target sequence spans from positions 19925852 to 19925877. This shRNA has been designed to maintain an intermediate GC content of approximately 30 - 50%, with 44% in this variant, to avoid strong secondary structures. This sequence has off-targets in *Vespa mandarinia*, *Dolichoris vasculosae*, and *Ganaspis brasiliensis*, with one mismatch in each, and in *Vespa crabro* with one INDEL. This shRNA contains the standard UUCAAGAGA 9 nt loop (Brummelkamp, Thijn R et al. “A system for stable expression of short interfering RNAs in mammalian cells.” *Science* (New York, N.Y.) vol. 296, 5567 (2002): 550-3. doi:10.1126/science.1068999). Through secondary structure analysis performed by ViennaRNA, the free energy of the thermodynamic ensemble is -46.55 kcal/mol, with the frequency of the minimum free energy structure in the ensemble at 35.03%. The ensemble diversity is 2.48.

Source
This part was synthetically designed within the context of this project.

Design consideration
None.

shRNA TS3_Hairpin2: BBa_K5047025

Short description
DNA encodes a shRNA that binds to the chitin synthase gene of V. velutina.

Long description
This DNA sequence encodes a short hairpin RNA (shRNA) designed to target a specific region within the chitin synthase gene of the Asian hornet *Vespa velutina*. The target region is located on an exon common to all isoforms of the chitin synthase gene (GeneID: 124951980, Genbank XM_047501172.1) within the genomic coordinates NC_062188.1 19915311-19930067 of the iVesVel2.1 genome (GCF_912470025.1). The specific target sequence spans from positions 19925852 to 19925877. This shRNA has been designed to maintain an intermediate GC content of approximately 30 - 50%, with 44% in this variant, to avoid strong secondary structures. This sequence has off-targets in *Vespa mandarinia*, *Dolichoris vasculosae*, and *Ganaspis brasiliensis*, with one mismatch in each, and in *Vespa crabro* with one INDEL. Upon testing in human embryonic kidney 293T cells using a luciferase assay, Schopman Nick C T et al. “Optimization of shRNA inhibitors by variation of the terminal loop sequence.” *Antiviral Research* vol. 86.2 (2010): 204-11. doi: 10.1016/j.antiviral.2010.02.320 reported that shRNAs based on the mir-17 and mir-25 loops exhibited the strongest target repression, approximately 10-fold stronger than the 9-nt loop. This shRNA contains the optimized UGAUAUGUGCA loop (Schopman Nick C T et al. *Optimization of shRNA inhibitors by variation of the terminal loop sequence* Antiviral research vol 86.2 2010 204-11 doi: 10.1016/j.antiviral.2010.02.320). Through secondary structure analysis performed by ViennaRNA, the free energy of the thermodynamic ensemble is -47.50 kcal/mol, with the frequency of the minimum free energy structure in the ensemble at 50.97%. The ensemble diversity is 0.77.

Source
This part was synthetically designed within the context of this project.

Design consideration
None.

shRNA TS3_Hairpin3: BBa_K5047026

Short description
DNA encodes a shRNA that binds to the chitin synthase gene of V. velutina.

Long description
This DNA sequence encodes a short hairpin RNA (shRNA) designed to target a specific region within the chitin synthase gene of the Asian hornet *Vespa velutina*. The target region is located on an exon common to all isoforms of the chitin synthase gene (GeneID: 124951980, Genbank XM_047501172.1) within the genomic coordinates NC_062188.1 19915311-19930067 of the iVesVel2.1 genome (GCF_912470025.1). The specific target sequence spans from positions 19925852 to 19925877. This shRNA has been designed to maintain an intermediate GC content of approximately 30 - 50%, with 44% in this variant, to avoid strong secondary structures. This sequence has off-targets in *Vespa mandarinia*, *Dolichoris vasculosae*, and *Ganaspis brasiliensis*, with one mismatch in each, and in *Vespa crabro* with one INDEL. Upon testing in human embryonic kidney 293T cells using a luciferase assay, Schopman Nick C T et al. “Optimization of shRNA inhibitors by variation of the terminal loop sequence.” *Antiviral Research* vol. 86.2 (2010): 204-11. doi: 10.1016/j.antiviral.2010.02.320 reported that shRNAs based on the mir-17 and mir-25 loops exhibited the strongest target repression, approximately 10-fold stronger than the 9-nt loop. This shRNA contains the optimized CCUCUCAACACUGG loop (Schopman Nick C T et al. *Optimization of shRNA inhibitors by variation of the terminal loop sequence* Antiviral research vol 86.2 2010 204-11 doi: 10.1016/j.antiviral.2010.02.320). Through secondary structure analysis performed by ViennaRNA, the free energy of the thermodynamic ensemble is -50.91 kcal/mol, with the frequency of the minimum free energy structure in the ensemble at 51.19%. The ensemble diversity is 0.77.

Source
This part was synthetically designed within the context of this project.

Design consideration
None.

shRNA TS4_Hairpin1: BBa_K5047027

Short description
DNA encodes a shRNA that binds to the chitin synthase gene of V. velutina.

Long description
This DNA sequence encodes a short hairpin RNA (shRNA) designed to target a specific region within the chitin synthase gene of the Asian hornet *Vespa velutina*. The target region is located on an exon common to all isoforms of the chitin synthase gene (GeneID: 124951980, Genbank XM_047501172.1) within the genomic coordinates NC_062188.1 19915311-19930067 of the iVesVel2.1 genome (GCF_912470025.1). The specific target sequence spans from positions 19920482 to 19920507. This shRNA has been designed to maintain an intermediate GC content of approximately 30 - 50%, with 36% in this variant, to avoid strong secondary structures. This sequence has off-targets in *Vespa mandarinia* and *Vespa crabro* with one mismatch and INDEL, and in *Cerceris rybyensis* and *Anoplius nigerrimus* with two mismatches in each. This shRNA contains the standard UUCAAGAGA 9 nt loop (Brummelkamp, Thijn R et al. “A system for stable expression of short interfering RNAs in mammalian cells.” *Science* (New York, N.Y.) vol. 296,5567 (2002): 550-3. doi:10.1126/science.1068999). Through secondary structure analysis performed by ViennaRNA, the free energy of the thermodynamic ensemble is -41.04 kcal/mol, with the frequency of the minimum free energy structure in the ensemble at 35.37%. The ensemble diversity is 2.54.

Source
This part was synthetically designed within the context of this project.

Design consideration
None.

shRNA TS4_Hairpin2: BBa_K5047028

Short description
DNA encodes a shRNA that binds to the chitin synthase gene of V. velutina.

Long description
This DNA sequence encodes a short hairpin RNA (shRNA) designed to target a specific region within the chitin synthase gene of the Asian hornet *Vespa velutina*. The target region is located on an exon common to all isoforms of the chitin synthase gene (GeneID: 124951980, Genbank XM_047501172.1) within the genomic coordinates NC_062188.1 19915311-19930067 of the iVesVel2.1 genome (GCF_912470025.1). The specific target sequence spans from positions 19920482 to 19920507. This shRNA has been designed to maintain an intermediate GC content of approximately 30 - 50%, with 36% in this variant, to avoid strong secondary structures. This sequence has off-targets in *Vespa mandarinia* and *Vespa crabro* with one mismatch and INDEL, and in *Cerceris rybyensis* and *Anoplius nigerrimus* with two mismatches in each. Upon testing in human embryonic kidney 293T cells using a luciferase assay, Schopman Nick C T et al. “Optimization of shRNA inhibitors by variation of the terminal loop sequence.” *Antiviral Research* vol. 86.2 (2010): 204-11. doi: 10.1016/j.antiviral.2010.02.320 reported that shRNAs based on the mir-17 and mir-25 loops exhibited the strongest target repression, approximately 10-fold stronger than the 9-nt loop. This shRNA contains the optimized UGAUAUGUGCA loop (Schopman Nick C T et al. Optimization of shRNA inhibitors by variation of the terminal loop sequence, *Antiviral Research* vol. 86.2, 2010, 204-11, doi: 10.1016/j.antiviral.2010.02.320). Through secondary structure analysis performed by ViennaRNA, the free energy of the thermodynamic ensemble is -42.40 kcal/mol, with the frequency of the minimum free energy structure in the ensemble at 51.90%. The ensemble diversity is 0.78.

Source
This part was synthetically designed within the context of this project.

Design consideration
None.

shRNA TS4_Hairpin3: BBa_K5047029

Short description
DNA encodes a shRNA that binds to the chitin synthase gene of V. velutina.

Long description
This DNA sequence encodes a short hairpin RNA (shRNA) designed to target a specific region within the chitin synthase gene of the Asian hornet *Vespa velutina*. The target region is located on an exon common to all isoforms of the chitin synthase gene (GeneID: 124951980, Genbank XM_047501172.1) within the genomic coordinates NC_062188.1 19915311-19930067 of the iVesVel2.1 genome (GCF_912470025.1). The specific target sequence spans from positions 19920482 to 19920507. This shRNA has been designed to maintain an intermediate GC content of approximately 30 - 50%, with 36% in this variant, to avoid strong secondary structures. This sequence has off-targets in *Vespa mandarinia* and *Vespa crabro* with one mismatch and INDEL, and in *Cerceris rybyensis* and *Anoplius nigerrimus* with two mismatches in each. Upon testing in human embryonic kidney 293T cells using a luciferase assay, Schopman Nick C T et al. “Optimization of shRNA inhibitors by variation of the terminal loop sequence.” *Antiviral Research* vol. 86.2 (2010): 204-11. doi: 10.1016/j.antiviral.2010.02.320 reported that shRNAs based on the mir-17 and mir-25 loops exhibited the strongest target repression, approximately 10-fold stronger than the 9-nt loop. This shRNA contains the optimized CCUCUCAACACUGG loop (Schopman Nick C T et al. Optimization of shRNA inhibitors by variation of the terminal loop sequence, *Antiviral Research* vol. 86.2, 2010, 204-11, doi: 10.1016/j.antiviral.2010.02.320). Through secondary structure analysis performed by ViennaRNA, the free energy of the thermodynamic ensemble is -45.40 kcal/mol, with the frequency of the minimum free energy structure in the ensemble at 52.16%. The ensemble diversity is 0.77.

Source
This part was synthetically designed within the context of this project.

Design consideration
None.

Deletion of the RNAseIII from the E. coli genome: BBa_K5047035

Short description
The Escherichia coli strain K-12 substrain MG1655 has been genetically modified to remove the RNAseIII, ensuring that RNA molecules, such as shRNA, do not undergo fast degradation.

Long description
The Escherichia coli strain K-12 substrate MG1655 has been engineered to lack the RNAseIII, the enzyme responsible for degrading RNA molecules. By removing RNase, the strain allows for greater stability and preservation of RNA constructs, such as shRNAs. This modification might be particularly relevant for experiments involving RNA interference, where the stability of RNA molecules is critical for efficient RNA-mediated gene silencing.

• To delete the RNAseIII gene, we followed the Red®/ET® Recombination protocol (https://static.igem.org/mediawiki/2018/b/bf/T--Munich--pRED_ET-Ecoli_Gene_Deletion_Kit.pdf.pdf): we first replaced the genomic gene with a kanamycin resistance cassette flanked by FRT sites, then we removed the antibiotic resistance thanks to the FLP flippase, thereby leaving a unique FRT scar at the location of the gene deletion.

Source
Genomic source, Escherichia coli strain K-12 substrain MG1655

Design consideration
None.

Composite part of AGO: BBa_K5047039

Short description
This sequence allows the constitutive expression of the Argonaute protein in yeast.

Long description
This sequence is composed of three parts:

1. A constitutive promoter (pPGK1): derived from the Saccharomyces cerevisiae gene phosphoglycerate kinase 1, which is known to be active in yeast cells under most growth conditions containing glucose. (Chambers, A et al. “Transcriptional control of the Saccharomyces cerevisiae PGK gene by RAP1.” Molecular and cellular biology vol. 9,12 (1989): 5516-24. doi:10.1128/mcb.9.12.5516-5524.1989)
2. The argonaute (AGO) protein coding sequence: a key component of the RNA-induced silencing complex (RISC)
3. A terminator (tSIF2) derived from a gene in yeast (SIF2) which helps in stabilising the transcript and ensuring efficient gene expression.

The Argonaute protein is a key part of the RISC complex, which is absent in S. cerevisiae. This construct enables the introduction and functional expression of RNA interference machinery in this yeast species.

Source
The promoter comes from the pVW222 plasmid given to us by Prof. Serge Pelet (DMF, university of Lausanne). The protein coding sequence and the terminator were synthetically designed to ensure compatibility and optimal function in yeast.

Design consideration
We divided the protein coding sequence of AGO in 3 parts, that we ordered as g-blocks from IDT. The 3 g-blocks facilitate the handling, and assembly of long DNA sequences and were designed to have overlapping regions with each other and with the promoter and terminator.

Name of the parts
pPGK1 (Promoter)
AGO (Argonaute Protein Coding Sequence)
tSIF2 (Terminator)

Composite part of DICR: BBa_K5047040

Short description
This sequence allows the constitutive expression of the Dicer protein in yeast.

Long description
This sequence is composed of three parts:

1. Constitutive Promoter (pTEF1): Sourced from the Saccharomyces cerevisiae gene TEF1, this promoter drives continuous expression in yeast cells across various growth conditions. (Mumberg, D et al. “Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds.” Gene vol. 156,1 (1995): 119-22. doi:10.1016/0378-1119(95)00037-7)
2. DICR Protein Coding Sequence: Encodes the Dicer protein, a crucial component of the RNA-induced silencing complex (RISC), which is essential for the processing of double-stranded RNA into small interfering RNAs (siRNAs).(Tang, Kai-Fu, and Hong Ren. “The role of dicer in DNA damage repair.” International journal of molecular sciences vol. 13,12 16769-78. 7 Dec. 2012, doi:10.3390/ijms131216769)
3. Terminator (tSTE2): Derived from the yeast gene STE2, the terminator ensures proper transcription termination and stabilises the resulting mRNA transcript, facilitating efficient protein expression.

The Dicer protein is a key part of the RISC complex, which is absent in S. cerevisiae. This construct enables the introduction and functional expression of RNA interference machinery in this yeast species.

Source
The pTEF1 promoter originates from the pSP329 plasmid provided by Prof. Serge Pelet (DMF, University of Lausanne). Both the DICR coding sequence and the tSTE2 terminator were synthetically designed to ensure optimal compatibility and performance in yeast.

Design consideration
The DICR coding sequence was divided into two segments, ordered as g-blocks from IDT. It was designed with overlapping regions between g-blocks, the promoter, and the terminator facilitating the efficient assembly of the full genetic sequence.

Name of the parts
pTEF1 (Promoter)
DICR (Dicer Protein Coding Sequence)
tSTE2 (Terminator)

Basic part pTEF1: BBa_K5047036

Short description
This sequence encodes for a constitutive promoter pTEF1 from Saccharomyces cerevisiae.

Long description
pTEF1 is a constitutive promoter for translation elongation factor 1α in Saccharomyces cerevisiae. (Mumberg, D et al. “Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds.” Gene vol. 156,1 (1995): 119-22. doi:10.1016/0378-1119(95)00037-7) This promoter is known for its robust and stable expression, making it a preferred choice for applications that require consistent gene expression in yeast. To evaluate the strength of the pTEF1 promoter in comparison to other commonly used promoters, we constructed a series of plasmids encoding the red fluorescent protein mCherry under the control of different promoters: pTEF1 (BBa_K5047036), pADH1 (BBa_J63005), and pCYC1 (BBa_K124000). These plasmids were used to determine relative expression levels in yeast.

• Plasmid pDA95
• Origins of replication in E. coli
• AmpR
• URA3
• pTEF1, mCherry
• Plasmid pDA120
• Origins of replication in E. coli
• AmpR
• URA3
• pTEF1, mCherry
• Plasmid pDA122
• Origins of replication in E. coli
• AmpR
• URA3
• pTEF1, mCherry

Experimental Design
The plasmids were linearized using the restriction enzyme BstBI for targeted integration into the URA3 locus of the Saccharomyces cerevisiae strain W303. This method ensures that each colony would have a single, defined integration site (Wosika, Victoria et al. “New families of single integration vectors and gene tagging plasmids for genetic manipulations in budding yeast.” Molecular genetics and genomics : MGG vol. 291,6 (2016): 2231-2240. doi:10.1007/s00438-016-1249-1), enabling direct comparison of promoter activity.
Following transformation, the yeast cultures were incubated for 2 days at 30°C. After sufficient growth, 12 colonies were selected from each transformation plate for further analysis. These colonies were cultured in SD full medium, a non-fluorescent medium, to avoid background fluorescence from affecting measurements.
The fluorescence intensity of mCherry expression in the samples was measured using a plate reader. Fluorescence was normalised by the optical density (OD) of each sample to account for differences in cell density. This normalisation enabled accurate comparison of the expression levels driven by the three different promoters.

Fluorescence Measurement and Flow Cytometry Analysis
The samples with sufficient levels of fluorescence expression were further analysed using flow cytometry, which allows for a detailed quantification of fluorescence intensity at the single-cell level.

Results
The data obtained from these experiments are consistent with each other and demonstrate that pTEF1 drives significantly higher expression of mCherry compared to both pADH1 and pCYC1. This confirms that pTEF1 is a stronger promoter, making it ideal for experiments where high and constitutive expression of a target gene is required.

Figure 1: Analysis of the expression strength of three constitutive promoters (pPGK1, pTEF1, and pADH1) driving mCherry expression in yeast, measured via flow cytometry.

Figure 2: Analysis of the expression strength of three constitutive promoters (pPGK1, pTEF1, and pADH1) driving mCherry expression in yeast, measured via plate reader assay.

Conclusion
The pTEF1 promoter shows higher performance in driving gene expression in Saccharomyces cerevisiae when compared to pADH1 and pCYC1. This makes pTEF1 a valuable tool for experiments requiring strong and consistent gene expression in yeast.

Source
The pTEF1 promoter originates from the pSP329 plasmid provided by Prof. Serge Pelet (DMF, University of Lausanne).
All common methods such as PCR, agarose gel electrophoresis, miniprep, and bacterial transformation are described in the Experiments section of the UniLausanne 2024 wiki page, Link. Analysis of the expression strength of three constitutive promoters (pPGK1, pTEF1, and pADH1) driving mCherry expression in yeast, measured via plate reader assay.

Basic part tSIF2: BBa_K5047037

Short description
tSIF2 terminator ensures stable Argonaute expression in yeast by enabling proper transcript termination.

Long description
The tSIF2 terminator is a crucial regulatory element for the efficient expression of genes in Saccharomyces cerevisiae (yeast). Derived from the SIF2 gene, this terminator plays an essential role in transcriptional regulation, ensuring that gene expression is both accurate and effective. Its primary functions include:

• Transcript Stabilisation: The tSIF2 sequence facilitates proper termination of mRNA transcripts, ensuring that they are stabilized and protected from degradation. This allows for the preservation of mRNA integrity, which is essential for the continuous and efficient expression of the associated gene.
• Efficient Expression: By providing a clear and reliable termination signal for RNA polymerase, the tSIF2 terminator ensures that transcription halts at the correct point, preventing read-through into adjacent genes. This precise termination enhances the expression efficiency of downstream genes by promoting the production of stable, functional mRNA that is ready for translation.

Source
The tSIF2 terminator was provided by Prof. Serge Pelet (DMF, University of Lausanne).

Design consideration
None.

Composite part of pTEF1 + mCherry: BBa_K5047041

Short description
This sequence encodes for a constitutive promoter pTEF1 followed by the red fluorescent reporter mCherry for expression in Saccharomyces cerevisiae.

Long description
This composite part was created to test the strength of the pTEF1 promoter using an mCherry reporter fluorescence assay.
To evaluate the expression efficiency of the pTEF1 promoter, a plasmid was first constructed where mCherry was placed under the control of pTEF1. The fluorescence intensity resulting from this construct allowed for a direct quantification of the promoter’s strength, since mCherry expression correlates with the activity of pTEF1.
To benchmark pTEF1's performance, its activity was compared to two other well-characterised promoters: pADH1 (BBa_J63005) and pCYC1 (BBa_K124000). Both of these promoters are commonly used, and well documented on the iGEM registry. By comparing the fluorescence intensity of mCherry expression under these three different promoters, it was possible to rank their relative strengths and effectiveness in driving gene expression.

Source
The pTEF1 promoter originates from the pSP329 plasmid and the mCherry reporter originates from the pDA95 plasmid, both plasmids were provided by Prof. Serge Pelet (DMF, University of Lausanne).

Design consideration
None.

Name of the parts
pTEF1 (Promoter)
mCherry (Reporter)

Composite part of pADH1 + mCherry: BBa_K5047042

Short description
This sequence encodes for a constitutive promoter pADH1 (BBa_J63005) followed by the red fluorescent reporter mCherry for expression in Saccharomyces cerevisiae.

Long description
As the pADH1 constitutive promoter was already characterised, this composite part served as a comparison point to quantify the strength of the pTEF1 promoter.

Source
The pADH1 promoter originates from the pDA122 plasmid and the mCherry reporter originates from the pDA95 plasmid, both plasmids were provided by Prof. Serge Pelet (DMF, University of Lausanne).

Design consideration
None.

Name of the parts
pADH1 (Promoter)
mCherry (Reporter)

Composite part of pCYC1 + mCherry: BBa_K5047043

Short description
This sequence encodes for a constitutive promoter pCYC1 (BBa_K124000) followed by the red fluorescent reporter mCherry for expression in Saccharomyces cerevisiae.

Long description
As the pCYC1 constitutive promoter was already characterised, this composite part served as a comparison point to quantify the strength of the pTEF1 promoter.

Source
The pCYC1 promoter originates from the pDA120 plasmid and the mCherry reporter originates from the pDA95 plasmid, both plasmids were provided by Prof. Serge Pelet (DMF, University of Lausanne).

Design consideration
None.

Name of the parts
pCYC1 (Promoter)
mCherry (Reporter)