Difference between revisions of "Part:BBa M36907:Design"

Revision as of 06:34, 9 December 2015

Coding Sequence of IscA1 pigeon homologue (codon-optimized for humans)

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 325
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 325
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 325
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 325
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 325
1000
COMPATIBLE WITH RFC[1000]

Design Notes

Schematic of IscA Plasmid Design

While the plasmid design for this insert can be complicated, the coding sequence itself is quite simple. We only needed to remove the start codon in order to co-express a fluorescent protein, and modify two BbsII restriction enzyme sites by altering the third base pair of codons (wobble effect) at bp locations 75 (C->G) and 309 (A->G).

Our first plasmid design builds off of DNA 2.0’s mammalian transient expression vector pD669-AR. It begins with a CMV strong constitutive promoter, which has demonstrated strong expression in HEK-293T cells. Since we want IscA protein expression in all cells transfected with the gene, transcription of the IscA gene will always be switched “on.” We are dealing with eukaryotic cells, so the 5’ cap of the mRNA acts as the ribosome binding site. Next we insert the coding sequence of our IscA genes (two variants--IscA1/IscA2). Taking the amino acid sequence from the pigeon (columba livia) homologues of these IscA isoforms from NCBI, we codon-optimized the sequence for homo sapiens. The IscA1 coding sequence has been registered on the iGEM Registry of Standard Biological Parts as BBa_M36907 and the IscA2 coding sequence has been registered under BBa_M36908. More information about modifications we made and the actual sequences can be found at iGEM Registry. Each IscA gene is fused to RudolphRFP (Ex/Em = 553/570nm) by a P2A sequence for easy cleavage to monitor protein expression and confirm translation. No stop codon is necessary since a bovine growth hormone signal is present to terminate protein expression in eukaryotic cells.

The plasmid confers both puromycin and ampicillin resistance; though for mammalian cells, antibiotic selection would only be the focus of follow-up projects as we will approximate eukaryotic expression with transient transfections in our pilot study. Moreover, the enhancer element from simian virus 40 (not shown) overcomes transcriptional inefficiencies in eukaryotic cells. This further validates our cell line choice, since 293T cells are derived from 293 cells but stably express the SV40 large T antigen which can bind to SV40 enhancers of expression vectors to increase protein production. Lastly, a high copy-number origin of replication (pUC, >500 copies) was used to maximize the amount of plasmid replicated, to ensure that each cell receives the magnetic receptor (although not critical for eukaryotic expression).

Source

The predicted IscA amino acid sequence was obtained from NCBI (http://www.ncbi.nlm.nih.gov/protein/XP_005508102.1), and codon-optimized for humans with IDT's tools.

References

https://www.idtdna.com/CodonOpt http://www.ncbi.nlm.nih.gov/gene/102090777

@@ Line 7: / Line 7: @@
 ===Design Notes===
-[[File:IscAGraphic.png|200px|thumb|left|alt text]]
+[[File:IscAGraphic.png|200px|thumb|left|Schematic of IscA Plasmid Design]]
 While the plasmid design for this insert can be complicated, the coding sequence itself is quite simple. We only needed to remove the start codon in order to co-express a fluorescent protein, and modify two BbsII restriction enzyme sites by altering the third base pair of codons (wobble effect) at bp locations 75 (C->G) and 309 (A->G).