Difference between revisions of "Part:BBa K3447004"

Revision as of 18:41, 27 October 2020

kanR

The kan^R gene is a 816 bp gene encodes a 271 amino acids’ ~31 kDa protein, which is resistant to kanamycin antibiotics, which is a class of antibiotics often used in molecular cloning.

• The characterization is done by adding a promoter BBa_J23100, see details in BBa_K3447108.

Introduction

Aminoglycoside antibiotics

The antibacterial mechanism of aminoglycoside antibiotics works by interfering with the accuracy of translation and the subsequent accumulation of error proteins. The misfolded protein that appears in the bacterial cell membrane increases the permeability of the drug, resulting in an increase in the concentration of intracellular aminoglycoside antibiotics, which plays a key role in sterilization and post-antibiotic effects. The ingestion of aminoglycoside antibiotics by bacteria is essential for their biological activity. The penetration of aminoglycoside antibiotics into bacterial cells requires three steps. The first is an energy-independent step, followed by two energy-dependent steps. Ribosomes and the respiratory chain that bind to the cell membrane are two essential substances for the accumulation of aminoglycoside antibiotic molecules in cells. That’s why anaerobic bacteria lacking an electron transport system are resistant to aminoglycoside antibiotics, such as enterococcus can resist low concentrations of amino sugar antibiotics. When aminoglycoside molecules come into contact with bacterial cells, polycationic antibiotic molecules bind to the anionic compounds of Gram-negative bacteria on the cell surface, such as lipopolysaccharides, phospholipids, and outer membrane proteins. The result of binding to the anionic site on the outer membrane is that the bridging divalent cations adjacent to the lipopolysaccharide molecule are displaced, resulting in increased permeability, forming "self-promoting absorption", and penetrating aminoglycoside molecules into the cytoplasmic space. The next process is called energy-dependent process I. In state I, only a small number of antibiotic molecules cross the cytoplasmic membrane, and a small number of molecules reaching the cytoplasm cause protein synthesis errors. The insertion of the incorrectly translated membrane protein destroys the integrity of the cytoplasmic membrane and initiates the next "energy-dependent process" Ⅱ". In this state, a large amount of amino sugars are transported across the injured cell plasma membrane. The higher the concentration of amino sugar, the faster the II state will be turned on. The abnormal protein in the injured lipid membrane is conducive to the transport of more antibiotic molecules, which enhances the interference with normal protein synthesis, causing greater damage to the cell membrane, forming an "autocatalytic type" to accelerate the absorption of antibiotics, and ultimately leading to cell death.

The molecular mechanism

The fidelity of the bacterial protein synthesis process is maintained by "monitoring" the base pairing between the mRNA codon and the tRNA anticodon at the ribosome coding site. The binding of ribosomes to the recognizable tRNA causes a conformational change in the coding site of ribosomal RNA, thus forming a signal to accelerate translation. The A1492 and A1493 adenylate residues contained in the inner loop of the 30S ribosomal subunit 16S rRNA constitute a major part of the bacterial coding site. The conformational flexible residues A1492 and A1493 are in contact with the minor groove of the codon-inverse codon hybrid. It is speculated that this is a geometric structure that "senses" the base pairing between mRNA and tRNA.

Figure 1. Main principles of the molecular mechanism of aminoglycoside antibiotics

Aminoglycoside antibiotics specifically bind to bacteria. The RNA coding site increases the probability of translation errors, and locks the residues A1492 and A1493 to interact with the mRNA-tRNA hybrid in a misplaced conformation. This leads to a decrease in the discrimination of unrecognizable tRNAs, thereby reducing translation accuracy Therefore, amino sugars can interfere with the coding process and eventually cause cell death, and the clinical effect of aminoglycoside antibiotics is reduced due to the modification of aminoglycoside antibiotics by bacterial resistance enzymes.

• Kanamycin is a typical aminoglycoside antibiotic.
  
  

Usage and Biology

Kanamycin is an aminoglycoside antibiotic that is an inhibitor of protein biosynthesis. It binds to 30S ribosomes to cause misreading of mRNA codes. If an enzyme that destroys kanamycin is produced in the bacteria, it can become a resistant strain. Plasmids resistant to kanamycin are often used as selection genes or marker genes in molecular cloning, produced by Streptomyces kanamyceticus, and the corresponding resistance gene is kan^R.

The kan^R gene controls the synthesis of kanamycin resistance protein. We will use kan^R to give engineered strains resistance for kanamycin.

The kan^R gene is a relatively common type of resistance gene in molecular cloning, and it is also very common in commercial plasmids. In the process of constructing plasmids, we will choose kanamycin-resistant vectors, so we no longer too much detail on its mechanism of action.

Characterization

• See more details in BBa_K3447108.
  

To verify the construction of Kan^R, the digestion and agarose gel electrophoresis were performed by a standard protocol (Fig. 2A). Compared with the control group, bacteria constructed with Kan^R show more than 10 times the resistance to kanamycin (Fig. 2B). Zone of inhibition test also shows that our Kan^R works well with 0.1% kanamycin added (Fig. 2C).

Fig. 2 Kan^R has resistance of kanamycin. (A) Digestion and electrophoresis of Kan^R. (B) Functional verification of Kan^R. E. coli DH5α was transformed with the designed plasmid, cultured overnight, and diluted OD₆₀₀ to 0.02. Measurement at the indicated time with 0.1% kanamycin added. The experiment was performed three times in triplicate. *, P < 0.05 compared with respective control. (C) Zone of inhibition test. 50 μL suspension was spread over the face of LB agar plate, on which a hole was bored for 0.1% kanamycin added. Incubate at 37℃ for 12 hrs. The plasmid was transformed into E. coli DH5α, cultured overnight, and diluted OD600 to 0.02.

Design

Design Notes

We added some synonymous mutations to avoid part rules.

Source

The gene is derived from pGlt-Kan vector, we found this sequence data in the GenBank.

Sequence and Features