Part:BBa_K4144012

An improved RBS to reduce the leakage expression of promoter Pxyl

Several point mutations were introduced into this part in order to reduce the leakage expression of target protein

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]

BBa_K4144012 part page

Purpose

We used a sequence consisting of Pxyl, RBS（BBa_K143021）and pet8p to express the SAMe transporter in our previous experiments of production module, however, we found that the leakage of promoter was severe. Thus, we are trying to change the RBS sequence in order to reduce its strength. BBa_K4144012 is the weak RBS improved on the basis of BBa_K143021.

 

Characterization

To engineer an RBS with a lower efficiency, we looked up the designed strategy on the internet, and found key factors governing the binding efficiency of the RBS with the ribosome.

       Considering that the purpose is to lower the transcription efficiency, we reversed those factors that would increase the bind efficiency. For example, we found that the optimal distance between the RBS and the stat codon is around 9bp, so, we decreased this distance to around 4. As shown in Figure1.1.

Figure 1.1 Sequence and the design strategy of the RBS, the one on the bottom is the original sequence

       We designed the optimized RBS(BBa_K4144012) according to those criteria described, and we used site-directed mutagenesis protocol to introduce these mutations into the RBS.

       After this, we transformed the site mutagenized plasmid into E. coli, picked out single transformed colony, and incubated it using 5ml of LB media under 37 ℃ overnight, and measured the fluorescence intensity using microplate reader.

After optimizing the RBS, we performed validation experiments together with the original RBS. We linked the two RBS to the Pxyl and GFP, respectively, and after the plasmid was successfully transformed and the engineered bacteria was cultured for a period of time, we performed fluorescence assays with an enzyme marker to compare the performance of BBa_K143021 and BBa_K4144012 with blank plate and DH5α used as blank controls.

Figure 1.2 Original data and the bar chart generated using this data, describing the fluorescence intensity of the bacteria.

       As the figure shown above, the fluorescence expression of bacteria containing plasmid with BBa_K4144012 is significantly lower than that of plasmid with BBa_K143021. We believe that this optimized RBS will facilitate in the reduction of the leaking expression of the promoter pXyl, due to the lower expression initiated using BBa_K4144012. However, due to the lack of time, we didn’t have the opportunity of placing this construct in the bacteria, and use the fluorescence microscope to check the expression of the protein.

 

Reference

[1] Joshua S. Klein, Siduo Jiang, Rachel P. Galimidi, Jennifer R. Keeffe, Pamela J. Bjorkman, Design and characterization of structured protein linkers with differing flexibilities, Protein Engineering, Design and Selection, Volume 27, Issue 10, October 2014, Pages 325–330,  https://doi.org/10.1093/protein/gzu043https://doi.org/10.1093/protein/gzu043