Part:BBa_I732021

Template for Building Primer Family Member

By means of PCR and molecular cloning, we come to possess the promoters that will serve as templates for the promoter family. There are several conservative sequences in the promoters, for instance, the -35 box, the -10 box, +1 start. Besides these conservative essence of all the transcriptable genes, we add two non-sense sequences, one on each side of the conservative region. The non-sense sequences were randomly produced, yet once set, they will never change again. They have three main characters:

1. They will never present in complicated structures;
2. They will never include the restriction enzyme cutting sites that will be involved in the whole study;
3. They will never include the recognition sites of RNA Polymerases and those of either of the two repressors.

In addition, we have introduced two operators with a pair of forward and backward primers. For example, U097O26+D062O16 indicates that this part has an operator O26 on the 97th base site upstream and an operator O62 on the 62th base site downstream. At the same time, we have also intruduced two cleavage sites outside each of the two operators, respectively.

P_template1 serves as the truss for PCR cloning. By applying 7 different pair of primers, we have got 49 different promoters. Then we made them digested and loaded into a vector that has already possessed a lacZ-alpha fragment. In this way, we can read from the color of the clones on the plate whether the inserted promoter works or not.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 19
  

Contribution by Team ZJFH-NANJING 2023

        For the Contribution, we supplemented the experimental characteristics of the part elements in Escherichia coli (BBa_I732021, BBa_K2213000). These studies involve two main aspects: construction of a T7RNAP library based on the PlacUV5 mutant and replacing the lacUV5 promoter with different expression strengths to alleviate the burden on the host organism. These data were added to the corresponding BioBricks.

Construction of T7 RNAP expression library

        lacUV5 promoter is mostly utilized for the efficient expression of T7RNAP, further combined with the T7 promoter to achieve high-level expression of recombinant proteins or target genes (Fig.1). In our investigation, we initially employed the pET system for expressing xylose reductases from different sources to produce xylitol. However, achieving high-level expression of T7RNAP does not necessarily enhance the yield of the target gene. On the contrary, it is essential to consider the physiological characteristics of the target gene, aiming to balance the expression relationship between the two. This approach allows for obtaining an optimal expression intensity ratio that not only enhances the expression of the target gene but also reduces the burden on the growth host of E.coli [1].

Fig.1: Schematic Diagram of pET Expression System

        Therefore, based on the obtained promoter library, we assembled the T7 RNAP onto plasmids with different strengths of promoters for testing. The resulting pET expression system can achieve the production of various intensities of combinations and can be applied to different types of E.coli (such as DH5α, MG1655, etc.). Here, we chose DH5α as a representative strain for application. Through qPCR analysis of transcriptional levels, we found that by optimizing in this manner, we obtained a T7RNAP library with different intensities, and the transcriptional levels showed a positive correlation with the previously measured fluorescence intensities (Fig.2).

Fig.2: Relative transcription levels of T7 RNAP

        All of these may be helpful to other teams and we hope it will make some contribution to the iGEM community.

Reference

        [1] Zhang ZX, Nong FT, Wang YZ, Yan CX, Gu Y, Song P, Sun XM. Strategies for efficient production of recombinant proteins in Escherichia coli: alleviating the host burden and enhancing protein activity. Microb Cell Fact. 2022 Sep 15;21(1):191.