Difference between revisions of "Part:BBa K3105666"
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in <I>Escherichia coli</I> when expressed in a low copy plasmid (pSB3K3), as shown in <b>Figure 3 & 4</b>. | in <I>Escherichia coli</I> when expressed in a low copy plasmid (pSB3K3), as shown in <b>Figure 3 & 4</b>. |
Revision as of 13:19, 18 October 2019
AsPink with ribosome standby site upstream of RBS
Group: iGEM19_Uppsala_Universitet
Improvement
The iGEM Team Uppsala 2019 added the ribosome standby site upstream of the RBS in Part: BBa_K1033926 by inverse PCR mutagenesis. This improved part BBa_K3105666 has higher expression of the chromoprotein AsPink than the original part BBa_K1033926 in Escherichia coli when expressed in a low copy plasmid (pSB3K3), as shown in Figure 3 & 4.
The improved part increased expression because there is an addition of a ribosome standby site (RSS). This RSS is situated between the promoter and ribosome binding site and works as an unstructured flanking region before the RBS in mRNA. When the RBS is inaccessible because it forms a stem-loop structure, this flanking region can serve as a site on which an initiation complex of the ribosome can assemble.
In Wagner’s paper, 9 CA repeats in the RBS standby site is shown to be able to dramatically upregulate protein expression by almost 10-fold. So, 9 CA repeats were chosen to be the inserted RSS to create a new part: BBa_K3105666 (Maaike Sterk et al., 2018)
The plasmid pSB1C3_BBa_K1033926 from the 2019 distribution kit was cut with EcoRI & PstI and then ligated into pSB3K3. A low-copy number plasmid was chosen since it will clearly exhibit an increase in protein expression. After ligation and transformation, construct: pSB3K3_K1033926 (pSB3K3_aspink) was built. Inverse PCR mutagenesis was then used on the above construct to insert 9 CA repeats as RSS with primers shown in Figure 1. By inserting the RSS, the RBS is expected to be released from stem-loop as illustrated in Figure 2. (The mRNA secondary structure prediction was done in CLC Main Workbench)
A significant color difference between the Escherichia coli (E. coli) colonies transformed with pSB3K3_aspink and pSB3K3_RSS_aspink can be identified by the naked eye, as shown in Figure 3.
Then a colorimetric assay was performed to measure the color intensity: E.coli colonies, picked from the plate shown in Figure 3 were cultured in liquid LB medium overnight. Then cells were lysed with lysozyme and Triton®X-100. After centrifugation, the supernatant was taken for measurement in the spectrophotometer at a wavelength of 572 nm. The result, as shown in Figure 4, indicated that there is a higher expression of AsPink in colonies transformed with the improved part (pSB3K3_RSS_aspink) compared to the control (pSB3K3_aspink).
In conclusion, 9 CA repeats insertion in the RBS standby site of the part K1033926 caused an upregulation of the expression of AsPinkin the low-copy plasmid pSB3K3.
Reference:
Sterk, M., Romilly, C., & Wagner, E. H. H. (2018). Unstructured 5’-tails act through ribosome standby to override inhibitory structure at ribosome binding sites. Nucleic Acids Research, 46(8), 4188-4199. doi:10.1093/nar/gky073
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]