Difference between revisions of "Part:BBa K4344007"

Latest revision as of 14:03, 12 October 2022

EcoRI-HSV VP5-fwd.

Part of our primer set used for inserting Kozak-sequence together with following parts: BBa_K4344007 (EcoRI-HSV UL19), BBa_K4344018 (HindIII-HSV UL19-rev.), BBa_K4344045 (pRK5-Kozak-fwd.). EcoRI-HSV-UL19 primer was not used in the final project, but still works.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 5
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 5
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 5
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 5
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 5
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K4344007 short</partinfo>
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+Part of our primer set used for inserting Kozak-sequence together with following parts: BBa_K4344007 (EcoRI-HSV UL19), BBa_K4344018 (HindIII-HSV UL19-rev.), BBa_K4344045 (pRK5-Kozak-fwd.). EcoRI-HSV-UL19 primer was not used in the final project, but still works.
 <!-- Add more about the biology of this part here
 ===Usage and Biology===
+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.
+The expression of the HSV-<i>UL19</i> 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 <span class="scientific-src">(Alekhina & Vassilenko, 2012)</span>. This isn’t always the first AUG, but the first AUG with a surrounding  Kozak-sequence <span class="scientific-src">(Dunston <i>et al.</i>,  2004)</span>. This sequence was missing in our plasmid initially. Either it was removed previously by the research group that provided us with this plasmid backbone or this specific version didn’t have one to begin with. The pRK5 has not one single version, but different versions of the plasmid have been published and are available to purchase. After cross referencing with the supplier Addgene, we noticed that there are multiple proposed plasmid vector maps (see notes at Addgene plasmid 3944).
+In our plasmid the CMV promoter from the human cytomegalovirus is used.. It is a transiently expressed promoter with high level of expression and good long-term efficiency in most cell types <span class="scientific-src">(Damdindorj <i>et al.</i>,  2014)</span>, therefore appropriate to be utilised in cell culture for investigating proof-of-principle or proof-of-concept studies in a simple but efficient manner.
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