Difference between revisions of "Team:Valencia UPV/Parts"

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<h2>Parts</h2>
 
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<p>Know our parts</p>
 
<p>Know our parts</p>

Revision as of 15:05, 16 September 2015

Valencia UPV iGEM 2015


Parts

Know our parts

Parts sent to the registry

Our team sent 14 Parts to the Registry:


Part NumberPart NameBackboneTypeLength
BBa_K1742000AsLOVpeppSB1C3Coding456
BBa_K1742001Enterotoxin LTBpSB1C3Coding414
BBa_K1742002Dronpa 145NpSB1C3Coding693
BBa_K1742003Dronpa 145KpSB1C3Coding741
BBa_K1742004PhiC31 Plant codon optimizedpSB1C3Coding1836
BBa_K1742005BxBI integrasepSB1C3Coding1693
BBa_K1742006BxBI Reporter (attB:T35S:attP:OmegaUTR)pSB1C3Reporter397
BBa_K1742007LTF LactoferrinpSB1C3Coding2079
BBa_K1742008PhiC31 Reporter (attP:T35S:aatB:OmegaUTR)pSB1C3Reporter412
BBa_K174200935S:BxbI:T35S - 35S:ReporterBxBI::GFP:T35SpSB1C3Device2964
BBa_K174201035S:LexABD-DronpaK:T35S - 35S:DronpaN-VP16:T35SpSB1C3Device3176
BBa_K174201135S:LacIBDBD-DronpaK:T35S - 35S:DronpaN-VP16:T35SpSB1C3Device2592
BBa_K174201235S:Gal4BD-DronpaK:T35S - 35S:DronpaN-VP16:T35SpSB1C3Device3610
BBa_K174201335S:PhiC31:T35S - 35S:ReporterPhiC31::GFP:T35SpSB1C3Device5508

BBa_K1742000 - AsLOVpep

AsLOVpep is a protein composed by the second LOV (light-oxigen-voltage) domain of Avena sativa phototropin and a C-terminus peptide tag for ePDZ recognition. This part was used to complete a blue light-induced switch (along with BBa_K1470005) present in the first and second modules of our biological circuit.

BBa_K1742001 - Enterotoxin LTB

The LTB is a heat-labile enterotoxin beta subunit from Escherichia coli. This DNA sequence underwent codon usage optimization for Nicotiana tabacum. LTB protein was used to test its expression levels in N. benthamiana leaves.

BBa_K1742002 - Dronpa 145N

Dronpa 145N is a green fluorescence protein that contains a K145N substitution. It is switched off upon illumination with cyan light and switched on with violet light. It is also capable to form heterodimers with Dronpa K (BBa_K1742003) upon violet light exposure. This DNA sequence underwent codon usage optimization for Arabidopsis thaliana. This part was used to test its potential as a transgene expression system.

BBa_K1742003 - Dronpa 145K

Dronpa 145K is the wild type protein used as a template to obtain Dronpa 145N (BBa_K1742002). This part was used to test its potential as a transgene expression system

BBa_K1742004 - PhiC31 Plant codon optimized

PhiC31 is a site-specific serine recombinase derived from the PhiC31 phage. The enzyme recognizes two different attachments sites called also attB and attP, and also excise a sequence flanked with attB and attP sites. This DNA sequence underwent codon usage optimization for Nicotiana benthamiana. We used PhiC31 as excisionase by flanking a T35S terminator with the recognition sites.

BBa_K1742005 - BxBI integrase

Bxb1 is a serine recombinase protein from Mycobateriophage Bxb1’s gp35 gen. This integrase is able to recognize two different sites, attP and attB. Depending on the position and sense of these sites bxb1 is able of excising or inverting. We used bxb1 as excisionase by flanking a T35S terminator with the recognition sites.

BBa_K1742006 - BxBI Reporter (attB:T35S:attP:OmegaUTR)

The part consists of Cauliflower Mosaic Virus T35S terminator flanked by the BxbI recognition sites attB and attP. It also contains an Omega UTR region. This part was used to test BxbI activity in N. benthamiana plants.

BBa_K1742007 - LTF Lactoferrin

This part codifies the complete Homo sapiens lactoferrin CDS.

BBa_K1742008 - PhiC31 Reporter (attP:T35S:aatB:OmegaUTR)

The part consists of Cauliflower Mosaic Virus T35S terminator flanked by the BxbI recognition sites attP and attB. It also contains an Omega UTR region. This part was used to test PhiC31 activity in N. benthamiana plants.

BBa_K1742009 - 35S:BxbI:T35S - 35S:ReporterBxBI::GFP:T35S

This device consists of two transcriptional unit modules: the BxbI integrase (BBa_K1742005) driven by the constitutive promoter P35S and the reporter element (BBa_K1742006) that contains the recognition sites flanking a T35S terminator and a GFP protein as a gene marker. When BxbI is expressed, it recognizes the attB and attP sites, excising the terminator and provoking the transcription of GFP.

BBa_K1742010 - 35S:LexABD-DronpaK:T35S - 35S:DronpaN-VP16:T35S

This device for plants consists of two modules: A Dronpa K fused to the DNA binding domain LexA, and a second transcriptional unit that consists of Dronpa N fused to the transcriptional activator VP16. Upon illumination with violet light, it is produced a heterodimerizacion between Dronpa K and N, leading to the expression of a gene of interest driven by LexA operator and a minimal promoter mini35S. This part was used to test the light-induced toggle switch functionality.

BBa_K1742011 - 35S:LacIBDBD-DronpaK:T35S - 35S:DronpaN-VP16:T35S

This device for plants consists of two modules: A Dronpa K fused to the DNA binding domain LacI, and a second transcriptional unit that consists of Dronpa N fused to the transcriptional activator VP16. Upon illumination with violet light, it is produced a heterodimerizacion between Dronpa K and N, leading to the expression of a gene of interest driven by LacI operator and a minimal promoter mini35S. This part was used to test the light-induced toggle switch functionality.

BBa_K1742012 - 35S:Gal4BD-DronpaK:T35S - 35S:DronpaN-VP16:T35S

This device for plants consists of two modules: A Dronpa K fused to the DNA binding domain Gal4, and a second transcriptional unit that consists of Dronpa N fused to the transcriptional activator VP16. Upon illumination with violet light, it is produced a heterodimerizacion between Dronpa K and N, leading to the expression of a gene of interest driven by UAS operator and a minimal promoter mini35S. This part was used to test the light-induced toggle switch functionality.

BBa_K1742013 - 35S:PhiC31:T35S - 35S:ReporterPhiC31::GFP:T35S

This device consists of two transcriptional unit modules: the PhiC31 integrase (BBa_K1742004) driven by the constitutive promoter P35S and the reporter element (BBa_K1742008) that contains the recognition sites flanking a T35S terminator and a GFP protein as a gene marker. When PhiC31 is expressed, it recognizes the attP and attB sites, excising the terminator and provoking the transcription of GFP.


Improved parts

Valencia UPV team did also improve parts that already existed in the Registry:

  • BBa_K1470005: We obtained this part from the Registry. This part consists of the engineered PDZ domain, a crucial part of the light-inducible expression system. It is able to bind to the J-α helix of the Avena sativa LOV2 domain. We designed, sequence synthetized and domesticated the AsLOV protein in order to complete this system. The AsLOV domain is tagged with a small peptide in the C-terminus, which allows its recognition by ePDZ upon illumination with blue light. We used both parts to test their functionality as a light-inducible switch in N. benthamiana plants.
  • BBa_K1132009: This part that was request to the Registry consists of the PhiC31 integrase. It encodes a serine-type recombinase that mediates the sequence-specific recombination between two different attachment sites, called attB and attP, which share a 3 bp central region, where the crossover occurs. Our team did characterize the functionality of this part in N. benthamiana plants. To do so, we used PhiC31 as excisionase of our PhiC31-Reporter element (BBa_K1742008) coupled with a GFP tag. After transiently Agrobacterium-mediated transformation in plants, we could observe that a huge percentage of plant cells expressed GFP. See our PhiC31 recombinase module results.