Team:TecCEM/Basic Part
Parts
Basic Parts
BBa_K2246000
BSLA: siRNA production and reporting cassette
Composing Parts: Basic
This part is for producing any small interfering RNA (siRNA), it is produced attached to a mRNA for expressing blue chromoprotein as a reporter. Cassette designed for E.coli siRNA production. This part may express any siRNA sequence desired, as it includes a BamHI and HincII cut sites limiting the region where the siRNA sequence is to be inserted. Also include a blue chromoprotein coding region as a reporter of production of the mRNA. The region designed for siRNA insertion contains the AWD siRNA coding region in the format (sense, 9 bp loop, anti-sense).
This part is a universal expression cassette for small interfering RNA (siRNA) in E.coli. This cassette contains the coding sequence of amilCP Blue Chromoprotein (BBa_K592009) as a reporter of expression. This method, designed by team TecCEM 2017, is named BSLA, which stands for Blue chromoprotein, Sense, Loop, Antisense; as it is its structure. The desired siRNA must be produced with the format (BamHI cut-site, sense, loop, antisense ending with blunt end). It is recommended to synthetisize two DNA oligos with the previously metioned format, then align them. The cassette must be digested with BamHI and HincII, then ligated with the siRNA oligos. Finally the ligation is transformed into E.coli HT115 to maximize the production. This part comes with the insert for producing AWD siRNA (BBa_K2246002), and it must be removed for the production of other siRNA.
BBa_K2246001
T7 Terminator
Composing Parts: Basic
Widely used T7 terminator
Terminator for parts with T7 promoter (BBa_I712074), a promoter recognized by T7 RNApol, a highly active enzime for a high transcription rate (Canton B., 2008).
References: Canton B. (2008). Engineering the interface between cellular chassis and synthetic biological systems. Retrieved from https://s3-us-west-2.amazonaws.com/oww-files-public/4/43/Canton-PhD-BE-2008.pdf
BBa_K2246002
Awd siRNA
Composing Parts: Basic
siRNA sequence for silencing Abnormal Wing Disc gene in Diaphorina citri.
This sequence must be used as dsRNA, in this way it works as a small interfering RNA specific for Abnormal Wing Disc (Awd) gene in D.citri. This gene is involved in larvae growth and wing development, when silenced it has been shown to increase nymph mortality and lead to the development of damaged wings, therefore reducing psyllid's ability to fly, thus affecting its movility (El-Shesheny, Hajeri, El-Hawary, Gowda & Killiny, 2013). The selected target sequence, in order to maximize specificity, was choosen by comparing the sequence of this gene against several species, and an exclusive region from D.citri was selected. Then a BLAST was run in order to eliminate the possibility of hybridization with other species.
References: El-Shesheny I, Hajeri S, El-Hawary I, Gowda S, Killiny N .(2013). Silencing Abnormal Wing Disc Gene of the Asian Citrus Psyllid, Diaphorina citri Disrupts Adult Wing Development and Increases Nymph Mortality. PLoS ONE 8(5): e65392. doi:10.1371/journal.pone.0065392
BBa_K2246003
RacI siRNA
Composing Parts: Basic
siRNA sequence for silencing Rac GTPase-activating protein 1 (RacI) gene in Diaphorina citri.
This sequence must be used as dsRNA, in this way it works as a small interfering RNA specific for rac GTPase-activating protein 1 (RacI) gene in D.citri. This gene encodes a GTPase-activating protein (GAP) that is a component of the centralspindlin complex. This protein binds activated forms of Rho GTPases and stimulates GTP hydrolysis, which results in negative regulation of Rho-mediated signals. This protein plays a regulatory role in cytokinesis, cell growth, and differentiation. Among its related pathways are Immune System and Vesicle-mediated transport. In other insects attacked by parasitoids and pathogens, Rac1 up-regulation has been linked to hemocyte recruitment. In D.citri this gene is up-regulted, which suggest the overexpression affects the efective inmune response againts Candidatus liberibacter asiaticus (Vyas, Fisher, He, Nelson, Yin, Cicero, Willer, Kim, Kramer, May, Crow, Soderlund, Gang & Brown, 2015). The selected target sequence, in order to maximize specificity, was choosen by comparing the sequence of this gene against several species, and an exclusive region from D.citri was selected. Then a BLAST was run in order to eliminate the possibility of hybridization with other species.
References: Vyas M, Fisher TW, He R, Nelson W, Yin G, Cicero JM, et al. (2015) Asian Citrus Psyllid Expression Profiles Suggest Candidatus Liberibacter Asiaticus-Mediated Alteration of Adult Nutrition and Metabolism, and of Nymphal Development and Immunity. PLoS ONE 10(6): e0130328. doi:10.1371/ journal.pone.0130328
BBa_K2246004
WNT siRNA
Composing Parts: Basic
siRNA sequence for silencing protein wingless-like (WNT) gene in Diaphorina citri.
This sequence must be used as dsRNA, in this way it works as a small interfering RNA specific for wingless-like (WNT) gene in D.citri. Involved in wing and nymph development. The down expression of this gene leads to wingless psyllids, inducig mortality and affecting maturing (Vyas, Fisher, He, Nelson, Yin, Cicero, Willer, Kim, Kramer, May, Crow, Soderlund, Gang & Brown, 2015). The selected target sequence, in order to maximize specificity, was choosen by comparing the sequence of this gene against several species, and an exclusive region from D.citri was selected. Then a BLAST was run in order to eliminate the possibility of hybridization with other species.
References: Vyas M, Fisher TW, He R, Nelson W, Yin G, Cicero JM, et al. (2015) Asian Citrus Psyllid Expression Profiles Suggest Candidatus Liberibacter Asiaticus-Mediated Alteration of Adult Nutrition and Metabolism, and of Nymphal Development and Immunity. PLoS ONE 10(6): e0130328. doi:10.1371/ journal.pone.0130328
BBa_K2246005
SOD siRNA
Composing Parts: Basic
siRNA sequence for silencing superoxide dismutase [Cu-Zn] (SOD) gene in Diaphorina Citri
This sequence must be used as dsRNA, in this way it works as a small interfering RNA specific for superoxide dismutase [Cu-Zn] (SOD) gene in D.citri. Cell metabolism CSD1 is an important superoxide dismutase involved in detoxification of superoxide radicals, therefore it silencing result in an increase of mortality rate (Taning, Andrade, Hunter, Christiaens & Smagghe, 2016). The selected target sequence, in order to maximize specificity, was choosen by comparing the sequence of this gene against several species, and an exclusive region from D.citri was selected. Then a BLAST was run in order to eliminate the possibility of hybridization with other species.
References: Taning C., Andrade E., Hunter W., Christiaens O. & Smagghe G. (2016). Asian Citrus Psyllid RNAi Pathway –RNAi evidence. Scientific Reports. DOI: 10.1038/srep38082
BBa_K2246006
GFP siRNA
Composing Parts: Basic
siRNA sequence for silencing protein green flourescent protein (GFP) gene.
This sequence must be used as dsRNA, in this way it works as a small interfering RNA specific for green flourescent protein (GFP) gene. Designed to be used as negative control during RNA interference experiments. In previous studies involving RNA interferance in D.citri, a dsRNA for GFP was used as negative control, see Taning, Andrade, Hunter, Christiaens & Smagghe (2016), El-Shesheny, Hajeri, El-Hawary, Gowda & Killiny (2013) and Yu, Gowda & Killiny (2017).
References: El-Shesheny I, Hajeri S, El-Hawary I, Gowda S, Killiny N .(2013). Silencing Abnormal Wing Disc Gene of the Asian Citrus Psyllid, Diaphorina citri Disrupts Adult Wing Development and Increases Nymph Mortality. PLoS ONE 8(5): e65392. doi:10.1371/journal.pone.0065392
Taning C., Andrade E., Hunter W., Christiaens O. & Smagghe G. (2016). Asian Citrus Psyllid RNAi Pathway –RNAi evidence. Scientific Reports. DOI: 10.1038/srep38082
Yu X., Gowda S. & Killiny N. (2017). Double-stranded RNA delivery throughsoaking mediates silencing of the muscleprotein 20 and increases mortality to the Asiancitrus psyllid, Diaphorina citri. Pest Management Science. DOI 10.1002/ps.4549
BBa_K2246007
RacI BSLA
Composing Parts: Basic
Device for the expression of RacI siRNA (BBa_K2246003)
This part is the cassette BSLA (BBa_K2246000) with the insertion of RacI siRNA coding sequence in the format (sense, loop, antisense), to express RacI siRNA (BBa_K2246003) on E.coli HT115.
BBa_K2246008
WNT BSLA
Composing Parts: Basic
Device for the expression of WNT siRNA (BBa_K2246004)
This part is the cassette BSLA (BBa_K2246000) with the insertion of WNT siRNA coding sequence in the format (sense, loop, antisense), to express WNT siRNA (BBa_K2246004) on E.coli HT115.
BBa_K2246009
SOD BSLA
Composing Parts: Basic
Device for the expression of SOD siRNA (BBa_K2246005)
This part is the cassette BSLA (BBa_K2246000) with the insertion of SOD siRNA coding sequence in the format (sense, loop, antisense), to express SOD siRNA (BBa_K2246005) on E.coli HT115.
BBa_K2246010
GFP BSLA
Composing Parts: Basic
Device for the expression of GFP siRNA (BBa_K2246006)
This part is the cassette BSLA (BBa_K2246000) with the insertion of GFP siRNA coding sequence in the format (sense, loop, antisense), to express GFP siRNA (BBa_K2246006) on E.coli HT115.
