Part:BBa_K3105666
AsPink with ribosome standby site upstream of RBS
AsPink (BBa_k1033906) with a ribosome standby site upstream of the RBS (J23110).
We added the ribosome standby site by inverse PCR mutagenesis and achieved higher expression than the original part in Escherichia coli when expressed in a low copy plasmid (pSB3K3).
The improved part(BBa_K3105666) has an increased expression of the chromoprotein AsPink, as compared to the part BBa_K1033926
The reason of improved part can increase expression is because of the ribosome standby site (RSS) that sits between the promoter and ribosome binding site. The RSS consists of a repeat of CA nucleotides. Since the RSS is downstream to the promoter it will be transcribed to RNA and this in turn will affect the secondary structure of the RNA. RSS is the unstructured flanking regions surrounding the RBS. And this site can enable formation of initiation complex even on an inaccessible RBS stem–loop.
In Wanger’s paper, 9 CA repeats in the RBS standby site is shown to be able to dramatically increase translation rates, almost 10-fold upregulation on protein expression. It was chosen to insert 9 CA repeats into the gene as a RSS. (Maaike Sterk et al., 2018)
The plasmid pSB1C3_BBa_K1033926 from the distribution kit was cut with SpeI and PstI then ligated into pSB3K3 which is a low-copy number plasmid. A low-copy number plasmid was chosen since it will clearly indicate an increase in protein expression. After ligation and transformation we got the plasmid pSB3K3_K10333926 (pSB3K3_aspink). Inverse mutagenesis was then done on the plasmid for inserting 9 CA repeats before the RBS, as in the Figure 1. Through this, the RBS is expected to release the stem loop as illustrated in Figure 2.
Figure 1. Primers for the 9 CA repeats insertion before RBS. The red characters is the overhang of each primer, this becomes the inserted nucleotides.
Figure 2. Prediction of mRNA secondary structure of original part: pSB3K3_aspink (A) and the RRS mutated part: pSB3K3_9CARSS_aspink (B).
Figure 3. Comparison of restreaking pSB3K3-aspink (Left) with pSB3K3_9CARSS_aspink (right)
A difference in color, as in Figure 3, can be identified by the naked eye that the improved part has a deeper pink color than umutated pBS3K3-aspink. This means that the insertion of a flanking region did improve the expression in the construct.
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 for the expression of aspink in 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]
| None |