Difference between revisions of "Part:BBa K4344028"
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A missing Kozak-sequence was inserted to enhance the translation of HSV <i>UL19</i> AA816:1148-6xHis via PCR. The HSV <i>UL19</i> fragment was amplified with PCR using Phusion 2x Mastermix with HF-Buffer (NEB), 0.5 µM pRK5-Kozak-fwd., HindIII-HSV <i>UL19</i>-rev. and roughly 1 ng of pEX-A258-HSV-<i>UL19</i>-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.
 
A missing Kozak-sequence was inserted to enhance the translation of HSV <i>UL19</i> AA816:1148-6xHis via PCR. The HSV <i>UL19</i> fragment was amplified with PCR using Phusion 2x Mastermix with HF-Buffer (NEB), 0.5 µM pRK5-Kozak-fwd., HindIII-HSV <i>UL19</i>-rev. and roughly 1 ng of pEX-A258-HSV-<i>UL19</i>-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.
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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.
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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.
  
  

Revision as of 14:05, 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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.