Part:BBa_K3105666

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_k1033906 by inverse PCR mutagenesis. This improved part BBa_K3105666 has higher expression of the chromoprotein AsPink than the original part BBa_k1033906 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)

Figure 1: Primer design for inverse PCR mutagenesis Primers for the 9 CA repeats insertion before RBS. The red characters are the overhangs of each primer, this becomes the inserted nucleotides.

Figure 2: Prediction of mRNA secondary structure . Original construct pSB3K3_aspink (A) RSS inserted construct: pSB3K3_RSS_aspink (B).

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.

Figure 3: Comparison of restreaked E. coli colonies Colonies transformed with pSB3K3-aspink (Left) and pSB3K3_RSS_aspink (right) on the same LB plate.

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).

Figure 4. Absorbance of DH5a lysate samples under 572 nm

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]