Difference between revisions of "Part:BBa K4344028"
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===Usage and Biology=== | ===Usage and Biology=== | ||
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| + | The primer pRK5-Kozak-fwd. (BBa_K4344045) contains this entirely sequence. It enables to insert this Kozak-sequence between the HSV-UL19 sequence (BBa_K4344020) together with the following parts: BBa_K4344007 (EcoRI-HSV UL19), BBa_K4344018 (HindIII-HSV UL19-rev.), BBa_K4344045 (pRK5-Kozak-fwd.). Future iGEM Teams can follow this set to insert the enhanced Kozak-sequence into eucaryotic expression vectors to run cellculture experiments based on human cells. It has a high value for therapeutic screenings. | ||
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Revision as of 15:36, 13 October 2022
Kozak Sequence (human RBS)
The expression of the HSV-UL19 mRNA was too low which is why we included an additional Kozak-sequence to our enhanced CMV-promoter. The Kozak-sequence is the conserved consensus sequence next to the promoter, surrounding the AUG for the start of transcription in eukaryotes. It plays a role in the binding of the ribosome complex to the AUG during initiation of transcription (Alekhina & Vassilenko, 2012). This isn’t always the first AUG, but the first AUG with a surrounding Kozak-sequence (Dunston et al., 2004). This sequence was missing in our plasmid initially.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
A missing Kozak-sequence was inserted to enhance the translation of HSV UL19 AA816:1148-6xHis via PCR. The HSV UL19 fragment was amplified with PCR using Phusion 2x Mastermix with HF-Buffer (NEB), 0.5 µM pRK5-Kozak-fwd., HindIII-HSV UL19-rev. and roughly 1 ng of pEX-A258-HSV-UL19-6xHis, the total reaction volume being 20 µL, for this purpose. The success of the PCR was evaluated on a 1.2 % TAE-Agarose gel stained with ethidium bromide. Successful PCR products were pooled and purified using the Qiagen PCR Clean-Up Kit. The concentration was measured using Nanodrop 2000.
Dunston, J. A., Hamlington, J. D., Zaveri, J., Sweeney, E., Sibbring, J., Tran, C., Malbroux, M., O'Neill, J. P., Mountford, R., & McIntosh, I. (2004). The human LMX1B gene: transcription unit, promoter, and pathogenic mutations. Genomics, 84(3), 565–576. https://doi.org/10.1016/j.ygeno.2004.06.002.
Alekhina, O. M., & Vassilenko, K. S. (2012). Translation initiation in eukaryotes: versatility of the scanning model. Biochemistry. Biokhimiia, 77(13), 1465–1477. https://doi.org/10.1134/S0006297912130056.