Team:AUC-EGYPT/Parts
We designed two sets of Toeholds: Series B-like and First Generation-Like (See Fig 1).
Figure 1 The two sets of Toeholds
We documented the parts on the regiestery of standard biological parts. See table 1 for the part name. Each Series B-like toehold has a corresponding First Generation-like part.
Attached is an excel sheet on the thermodynamic modeling data of each toehold.
Part of our circuit is CMV promoter, UAS Promoter, and GAL4BD-VP16. These parts were retrieved from the registery with the following names:
CMV: BBa_K2217024
UAS: BBa_K2575001
GAL4BD-VP16: BBa_K3242006
Part 1:
Basic part: pSeL120 promoter.
Description:
The baculovirus expression vector system (BEVS) is considered a powerful, robust and cost-effective technique for the production of eukaryotic proteins. Accordingly, it is important to enhance the production capabilities of the baculovirus vectors which can be done through the characterization of more effective promoters. As the host cells for the BEVS are insect cells, it was found that the Orf46 viral gene that codes for the polyhedron envelope protein (PEP), a structural protein that surrounds the polyhedra of the viral particles, is highly abundant in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Accordingly, Martínez-Solís, María et al. searched the possibility of using this regulatory sequence as a promoter for foreign gene expression using the BEVS. The hypothesized promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5). To generate the sequences for the tested promoter, the different fragments of the sequence upstream of the orf46 gene were added to recombinant baculovirus to test the GFP gene. Our focus will be on the pSeL 120 promoter which is 120 nt sequence upstream of the ATG start codon for the orf46 gene. In this sequence, the presence of a TAAG motif was revealed. This TAAG motif is in an AT rich region, which is a typical transcriptional initiation site of late and very late baculovirus promoters. As shown in the graphs retrieved from the paper, the highest promoter activity on average was achieved by the pSeL promoter as the GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh) promoter. The results were obtained through measuring the fluorescence at different time points after infection of Se301 (A), Sf21 (B), and Hi5 (C) cells. This promoter can be used to increase the production of several eukaryotic proteins while using the baculovirus system, but it was characterized for GFP production and reporter genes. Another improvement for this part is done through linking it to a polyhedrin promoter. Further details is found in our composite part (Part number).
Sequence:
GTTCAATAATCAATAAACCTCTCTATTATGCTTTGTAAATTTTTTATTTACTTTGTTGTCCATTGCTGATGATTTATTAAGTCTTTTGGCAAAAATTAAATTTTTGCTACAATATA
Part (2):
Composite part: Polyhedrin promoter + pSeL promoter.
Improved part: http://parts.igem.org/Part:BBa_K1734000
Description:
To further improve the functionality of the polyhedrin promoter (BBa_K1734000) and our newly added PSeL120 promoter (Part number). This composite part (part number) was created by combining the two promoters in tandem (polh-pSeL). The methodology in retrieved from Martínez-Solís, María et al. paper where they modified the polh-GFP vector by inserting the PCR resulting fragments of the pSeL120 promoter with the help of the the XhoI and AvrII restriction sites. The results for the expression levels resulting from this composite promoter is shown in figure (2 and 3). In figure (2), the relative fluorescence intensity, in different insect cell lines infected specifically: Se301 (A), Sf21 (B), and Hi5 (C) cells, was measured at with different time points after the infection of with different baculoviruses at a MOI of 5. In figure (3), another visual representation of the higher expression level of GFP caused by the polh-pSeL promoter in comparison to the separate promoters in sf21 cells. In conclusion, Increased productivity of the GFP was recognized in the case of the composite promoter, which is higher than the case of the pSeL or polh promoters alone.
Note figure 3 should be placed after figure 2.
Sequence:
Sequence of polh promoter followed by sequence for pSEL promoter.
GATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATT
GTTCAATAATCAATAAACCTCTCTATTATGCTTTGTAAATTTTTTATTTACTTTG
TTGTCCATTGCTGATGATTTATTAAGTCTTTTGGCAAAAATTAAATTTTTGCTACAATATA
Reference for first 2 parts:
Martínez-Solís, M., Gómez-Sebastián, S., Escribano, J. M., Jakubowska, A. K., & Herrero, S. (2016). A novel baculovirus-derived promoter with high activity in the baculovirus expression system. PeerJ, 4, e2183. https://doi.org/10.7717/peerj.2183
Contribution 2: Improvement for: Human phosphoglycerate kinase promoter (hPGK)
It’s important to mention one of the applications of hPGK promoter especially for the self-inactivating lentiviral vectors. We recommend using the hPGK promoter before the inserted gene of interest to the lentivirus vectors. The reason behind that is that this promoter helps in producing high expression levels of the gene of interest compared to the other non-viral promoters especially the mPGK. From the literature, we found that the murine phosphoglycerate kinase (mPGK) and human PGK (hPGK) were tested for their expression levels of canine CD18 through lentivirus vectors for gene therapy of canine leukocyte adhesion deficiency (CLAD) [1]. Although the hPGK-cCD18 LV vector was produced in low titers, but it had a transduction efficiency of 20% in CLAD CD34+ cells with normal levels of CD18 per cell. Also, the dogs treated with these vectors had levels of CD18+ neutrophils greater than 1.5% with 40%-50% of the neutrophils expressing normal levels of CD18 per cell and were able to survive for more than 2 years [1]. On the other hand, the mPGK-cCD18 LV vector can be produced in high titers and have higher transduction efficiency, but the treated dogs with this vector had very low levels of CD18 per cell, so none of them survived more than 7 months [1]. These results highlight the significant clinical difference between the two promotors, and they show further recommendation to use the hPGK promoter in the lentivirus vectors involved with gene therapy. Furthermore, we created a composite part (part number) involving the hPGK promoter to show how it can be used for high expressing rates in our modular baculovirus-mediated lentivirus vectors.
Improved part: http://parts.igem.org/Part:BBa_J176028
Reference:
Hunter, M. J., Zhao, H. M., Tuschong, L., Kapoor, V., Burkholder, T. H., Persons, D. A., & Hickstein, D. D. (2012). Human Versus Murine Phosphoglycerate Kinase (PGK) Promoter/Enhancer in Canine Leukocyte Adhesion Deficiency [Abstract]. Molecular Therapy, 20. doi:10.1016/s1525-0016(16)35945-7
Part 6:
Composite Part: Gag + Pol + RRE
Improved part: http://parts.igem.org/Part:BBa_K2703010
Description:
Through this composite part, we will be integrating the three basic parts (part no.1, no.2 and BBa_K2796017). This composite part will be an improvement on the part BBa_K2703010 as it should have contained the sequences for the gag and pol gene, but no sequences were uploaded for the part. Furthermore, we added the RRE to the end of this construct, so that it can be applied directly as part of one of the minimum four plasmids required for the production of third-generation lentiviruses (1). The gag/pol transcript required the RRE for its nuclear export. Otherwise, it will be inhibited because of its RNA secondary structures. Consequently, the vector structural and enzymatic proteins won’t be produced, and the vectors won’t assemble properly (1).
References:
Tareen, S. U., Nicolai, C. J., Campbell, D. J., Flynn, P. A., Slough, M. M., Vin, C. D., Kelley-Clarke, B., Odegard, J. M., & Robbins, S. H. (2013). A Rev-Independent gag/pol Eliminates Detectable psi-gag Recombination in Lentiviral Vectors. BioResearch open access, 2(6), 421–430. https://doi.org/10.1089/biores.2013.0037
Sequences for the basic parts of this composite part:
• Part 7: gag gene.
Description:
The gag gene is responsible for encoding capsid, matrix and nucleocapsid of the lentivirus vectors. Together with the pol gene and RRE form one of the essential four plasmids to create third generation lentiviruses with the minimal regions of the lentiviral genome. This part was only included in the design phase of the vectors and was not used in the lab, so there are no safety issues associated to it.
Sequence:
atgggcgcccgcgcctccgtgctgtccggcggcgagctggacaagtgggagaagatccgcctgc
gccccggcggcaagaagcagtacaagctgaagcacatcgtgtgggcctcccgcgagctgg
agcgcttcgccgtgaaccccggcctgctggagacctccgagggctgccgccagatcctggg
ccagctgcagccctccctgcaaaccggctccgaggagctgcgctccctgtacaacaccatcg
ccgtgctgtactgcgtgcaccagcgcatcgacgtgaaggacaccaaggaggccctggacaaga
tcgaggaggagcagaacaagtccaagaagaaggcccagcaggccgccgccgacaccggcaaca
actcccaggtgtcccagaactaccccatcgtgcagaacctgcagggccagatggtgcaccaggc
catctccccccgcaccctgaacgcctgggtgaaggtggtggaggagaaggccttctcccccgaa
gtcatccccatgttctccgccctgtccgagggcgccaccccccaggacctgaacaccatgctg
aacaccgtgggcggccaccaggccgccatgcagatgctgaaggagaccatcaacgaggaggcc
gccgagtgggaccgcctgcaccccgtgcacgccggccccatcgcccccggccagatgcgcgag
ccccgcggctccgacatcgccggcaccacctccaccctgcaagagcagatcggctggatgacc
cacaacccccccatccccgtgggcgagatctacaagcgctggatcatcctgggcctgaacaaga
tcgtgcgcatgtactcccccacctccatcctggacatccgccagggccccaaggagcccttccg
cgactacgtggaccgcttctacaagaccctgcgcgccgagcaggcctcccaggaggtaaagaac
tggatgaccgagaccctgctggtgcagaacgccaaccccgactgcaagaccatcctgaaggccct
gggccccggcgccaccctggaggagatgatgaccgcctgccagggcgtgggcggccccg
gccacaaggcccgcgtgctggccgaggccatgtcccaagtcaccaaccccgccaccatcatgatcc
agaagggcaacttccgcaaccagcgcaagaccgtgaagtgcttcaactgcggcaaggaggg
ccacatcgccaagaactgccgcgccccccgcaagaagggctgctggaagtgcggcaagg
agggccaccagatgaaagattgtactgagagacaggctaattttttagggaagatctg
gccttcccacaagggaaggccagggaattttcttcagagcagaccagagccaacagcccc
accagaagagagcttcaggtttggggaagagacaacaactccctctcagaagcaggagccg
atagacaaggaactgtatcctttagcttccctcagatcactctttggcagcgacccctcgtcacaa
• Part 8: pol gene.
Description:
The pol gene is responsible for encoding the enzymes protease, reverse transcriptase, and integrase of the lentivirus vectors. Together with the gag gene and RRE form one of the essential four plasmids to create third generation lentiviruses with the minimal regions of the lentiviral genome. This part was only included in the design phase of the vectors and was not used in the lab, so there are no safety issues associated to it.
Sequence:
agggaagatctggccttcccacaagggaaggccagggaattttcttcagag
cagaccagagccaacagccccaccagaagagagcttcaggtttggggaagag
acaacaactccctctcagaagcaggagccgatagacaaggaactgtatcct
ttagcttccctcagatcactctttggcagcgacccctcgtcacaataaagat
cggtggccagctgaaggaggccctgctggacaccggcgccgacgacaccgtgc
tggaggagatgaacctgcccggccgctggaagcccaagatgatcggcggcat
cggcggcttcatcaaagtccgccagtacgaccagatcctgatcgagatctgc
ggccacaaggccatcggcaccgtgctggtgggccccacccccgtgaacatc
atcggccgcaacctgctgacccagatcggctgcaccctgaacttccccatct
cccccatcgagaccgtgcccgtgaagctgaagcccggcatggacggccccaa
agtcaagcagtggcccctgaccgaggagaagatcaaggccctggtggagatc
tgcaccgagatggagaaggagggcaagatctccaagatcggccccgagaacc
cctacaacacccccgtgttcgccatcaagaagaaggactccaccaagtggcg
caagctggtggacttccgcgagctgaacaagcgcacccaggacttctgggag
gtgcagctgggcatcccccaccccgccggcctgaagcagaagaagtccgtga
ccgtgctggacgtgggcgacgcctacttctccgtgcccctggacaaggactt
ccgcaagtacaccgccttcaccatcccctccatcaacaacgagacccccggc
atccgctaccagtacaacgtgctgccccagggctggaagggctcccccgcca
tcttccagtgctccatgaccaagatcctggagcccttccgcaagcagaaccc
cgacatcgtgatctaccagtacatggacgacctgtacgtgggctccgacctg
gagatcggccagcaccgcaccaagatcgaggagctgcgccagcacctgctgc
gctggggcttcaccacccccgacaagaagcaccagaaggagccccccttcctgtg
gatgggctacgagctgcaccccgacaagtggaccgtgcagcccatcgtgctgccc
gagaaggactcctggaccgtgaacgacatccagaagctggtgggcaagctgaactgggcctcccaga
tctacgccggcatcaaagtccgccagctgtgcaagctgctgcgcggcaccaaggccctga
ccgaggtggtgcccctgaccgaggaggccgagctggagctggccgagaaccgcgagatc
ctgaaggagcccgtgcacggcgtgtactacgacccctccaaggacctgatcgccgagat
ccagaagcagggccagggccagtggacctaccagatctaccaggagcccttcaagaacct
gaagaccggcaaatacgcccgcatgaagggcgcccacaccaacgacgtgaagcagctgacc
gaggccgtgcagaagatcgccaccgagtccatcgtgatctggggcaagactcccaagttca
agctgcccatccagaaggagacctgggaggcctggtggaccgagtactggcaggccacct
ggatccccgagtgggagttcgtgaacaccccccccctggtgaagctgtggtaccagctgg
agaaggagcccatcatcggcgccgagaccttctacgtggacggcgccgccaaccgcgag
accaagctgggcaaggccggctacgtgaccgaccgcggccgccagaaggtggtgcccctga
ccgacaccaccaaccagaagaccgagctgcaggccatccacctggccctgcaagactccg
gcctggaggtgaacatcgtgaccgactcccagtatgcattgggcatcatccaggcccagcc
cgacaagtccgagtccgagctggtgtcccagatcatcgagcagctgatcaagaaggagaagg
tgtacctggcctgggtgcccgcccacaagggcatcggcggcaacgagcaggtggacaagctgg
tgtccgccggcatccgcaaggtgctgttcctggacggcatcgacaaggcccaggaggagcacga
gaagtaccactccaactggcgcgccatggcctccgacttcaacctgccccccgtggtggccaag
gagatcgtggcctcctgcgacaagtgccagctgaagggcgaggccatgcacggccaggtggact
gctcccccggcatctggcagctggactgcacccacctggagggcaaggtgatcctggtggccg
tgcacgtggcctccggctacatcgaggccgaggtgatccccgccgagaccggccaggagaccg
cctacttcctgctgaagctggccggccgctggcccgtgaagaccgtgcacaccgacaacggc
tccaacttcacctccaccaccgtgaaggccgcctgctggtgggccggcatcaagcaggagt
tcggcatcccctacaacccccagtcccagggcgtgatcgagtccatgaacaaggagctgaa
gaagatcatcggccaagtccgcgaccaggccgagcacctgaagaccgccgtgcagatggc
cgtgttcatccacaacttcaagcgcaagggcggcatcggcggctactccgccggcgagcg
catcgtggacatcatcgccaccgacatccagaccaaggagctgcagaagcagatcaccaag
atccagaacttccgcgtgtactaccgcgactcccgcgaccccgtgtggaagggccccgcca
agctgctgtggaagggcgagggcgccgtggtgatccaggacaactccgacatcaaggtggt
gccccgccgcaaggccaagatcatccgcgactacggcaagcagatggccggcgacgactgcgtggcctcccgccaggacgaggac
• Part 9: RRE.
Will be taken from http://parts.igem.org/Part:BBa_K2796017:Design
Part 11:
Basic part: Rev
Description: The rev gene codes for accessory proteins essential for the regulation of third generation lentivirus vectors. This gene was used in our composite part (part name).
Sequence:
atggcaggaagaagcggagacagcgacgaagacctcctcaaggcagtcagactcatcaagtttctctatcaaagcaacccacctcccaaccccgaggggacccgacaggcccgaaggaa
tagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatccttagcacttatctgggacgatctgcggagcctgtgcctcttcagctaccaccgcttgagagactta
ctcttgattgtaacgaggattgtggaacttctgggacgcagggggtgggaagccctcaaatattggtggaatctcctacagtattggagtcaggaactaaagaatag
Part 13:
Basic part: dsRed2
Improved part: http://parts.igem.org/Part:BBa_K1323009
Description: The dsRed gene (BBa_K1323009) was improved to produce a modified rapidly maturing tetrameric fluorescent protein (part number). The new fluorescent protein has minimal cytotoxicity, higher solubility, and exhibits strong and stable expression mammalian cells (1). It contains a N-terminus tag where cDNA can be added to form fusion protein and track them. However, our main usage for it is as a reporter gene prepared to be used in the lentivirus vector. We retrieved the dsRed2 gene and codon optimized it to be expressed in Spodoptera frugiperda (insect cells) to fit our baculovirus mediation expression system of the lentiviruses. More details can be found in (part collection link) and this gene is used in our composite part (part number) to increase its expression rates.
Reference:
Strack, R. L., Strongin, D. E., Bhattacharyya, D., Tao, W., Berman, A., Broxmeyer, H. E., Keenan, R. J., & Glick, B. S. (2008). A noncytotoxic DsRed variant for whole-cell labeling. Nature methods, 5(11), 955–957. https://doi.org/10.1038/nmeth.1264
Sequence:
atggcctcgtcggaaaacgtcataacggaatttatgcgtttca
aagttcgaatggaaggtaccgtcaatggacatgaatttgagat
agagggcgaaggtgaaggcaggccttatgaaggccataacacg
gtgaaattgaaagtgacaaagggcggaccgcttccgttcgcctg
ggatatactgtccccccagttccagtacggaagcaaggtatacgtta
aacaccctgcagacattccagattacaagaaattgagttttcctgagggttttaaatgggagcgtgtcatgaatttcg
aagacgggggcgtggcgacagtaactcaagactctagcctccaag
atggatgttttatttacaaagtaaagtttattggagttaattttcc
ctctgacggtcccgtcatgcaaaaaaaaaccatgggctgggaagcta
gtactgaaagactatacccgcgcgatggtgtgttgaaaggagaaac
gcataaagccttgaaactaaaggacggcggccattatttggtggaat
ttaaatcaatctatatggctaaaaaaccggttcaattaccaggtta
ttattacgtcgatgcgaagctggacatcacctcccacaacgaagatt
acactatcgtggaacaatacgaaagaacggagggccgccaccacttatt
ccAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCA
CAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCtctaa
Part 15:
Basic part: Tn7L
Sequence:
AACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGT
TCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACA
Part 16:
Basic part: Tn7R
Sequence: GAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTC
siRNA parts: BBa_K3626000 BBa_K3626001