Part:BBa_K1983012

tRNA-Phe (pheU) under DH10B promoter and terminator

Overview

This biobrick part codes the pheU gene from Escherichia coli for tRNA transporting L-phenylalanine with an upstream σ70 DH10B promoter and a terminator found after the tRNA. It is used to transport L-phenylalanine to the ribosome in the process of translation. The transcribed tRNA is processed by enzymes naturally occuring in E. coli to form a mature tRNA 76nt in length. The tRNA-Phe contains the anticodon GAA, thus it recognizes TTC as its sense codon to incorporate phenylalanine into the protein at protein translation.

Experiments and Results

Cloning

The received sequences were amplified using Uni-FW/RV primers and digested with EcoRI and PstI. The fragments were cloned into pSB1C3 vector digested with the same restriction enzymes. Transformant colonies were PCR-screened using VF2/VR primers and positive clone plasmids were sequenced prior to further usage.

Expression assays

Northern blot with two separate DNA probes complementary to the mature and immature forms of tRNA was performed in order to test the expression and the processing of the tRNA-Phe. This tRNA was tested with two different promoters: constitutive and σ70 DH10B. The results (Fig. 1) demonstrate that target RNA appears in cell in mature form, because it is only detected with a probe for mature tRNA, while no immature form is detected. In addition, the stronger promoter for the expression of tRNA appears to be the constitutive promoter. Because expression was tested in two E. coli strains the results in BL21 (DE3) strain overcomes the ones in ER2566.

Figure 1. Northern Blot of tRNA-Phe by comparing two different promoters. Red star indicates place of target tRNA. A Detection of tRNA-Phe with selective hybridization probe for mature tRNA-Phe in two different E. coli strains. B Detection of tRNA-Phe with selective hybridization probe for immature tRNA-Phe in two different E. coli strains.

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]