Part:BBa_K2150031

T7 promoter without RBS

This part is the T7 promoter without RBS. It has been reported that T7 promoter doesn't work with E.coli RNA polymerase, but only with T7 RNA polymerase(T7RNAP). However, we observed leakage of it in the absence of T7RNAP.

Improvement: Jilin_China 2019

Group:

Jilin_China 2019

Authors:

Yu Ma

Summary:

This year our team registered three mutated T7 promoters (BBa_K3078012,BBa_K3078013,BBa_K3078014) designed by Nie Z et al. Compared with the wild-type T7 promoter (BBa_K2150031), the expression intensity of BBa_K3078012 was 30% higher than that of wild-type T7 promoters.

Documention:

1. Design

T7 promoter is one of the most common promoters. But the intensity of the wild type T7 promoter often can’t meet our demand, so we mutated it and obtained an improved T7 promoter. According to previous studies, we selected the mutation site and constructed three mutated promoters (BBa_K3078012,BBa_K3078013,BBa_K3078014）based on the wild-type T7 promoter (BBa_K2150031) into the measurement vector (BBa_K3078100).

Table 1. Sequence of wild type and mutant T7 promoter.

Figure 1. Sequencing the plasmids.

2. Measurement

The promoter activity of wild-type and mutant T7 were measured through fluorescence intensity and normalized by OD₆₀₀. BBa_K2150031 and the mutated T7 promoter lack of T7 RNA polymerase, so we added IPTG to induce the expression of T7 RNA polymerase downstream of promoter lacUV5 of on the BL21(DE3) genome, as shown in Figure 2.

It can be seen from the figure that the promoter strength of mutant is higher than wild type.

Figure 2. The plasmid was transferred to E.coli BL21, cultured overnight, and diluted OD₆₀₀ 0.02. IPTG was added at OD₆₀₀ 0.3 to make the final concentration of IPTG reach 1mM to measure at the indicated time.

Contribution: New documentation from Evry_Paris-Saclay 2020

This part is the strong promoter from T7 bacteriophage (taatacgactcactata) with GGG at 3' end.

The T7 RNA polymerase initiates the transcription at the first guanidine of this stretch of three G and it was shown that +1 GGG is one of the best +1, +2 and +3 base combinations at the transcription initiation for enhanced promoter strength [1].

References

[1] Imburgio D, Rong M, Ma K, McAllister WT. Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants. Biochemistry (2000) 39, 10419-10430.

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]