Cloning of β-Lactamase Encoding Gene as the Initiation Approach in Providing High Quality Milk

DOI

10.2991/978-2-38476-261-3_10How to use a DOI?

Keywords

Antibiotics; Recombinant Plasmid; pLA230; Gene Cloning; PCR

Abstract

In silico exploration of several recombinant plasmids by previous study, informed that recombinant plasmid pLA230 is one of the plasmids harboring potent β-lactamase encoding gene producing enzyme that possesses specific activity to degrade penicillin antibiotics and its derivatives. The known target gene sequence in the recombinant plasmid pLA230 can facilitate the DNA isolation process, as source of β-lactamase genes that are more specific than gene isolation from wildtype organisms. Based on the information related to the potential of β-lactamase gene source pLA230, it can then be used as the basis to conduct direct gene exploration through isolation of β-lactamase gene from plasmid pLA230 and validate the nucleotides sequence via sequencing method. This study aims to obtain β-lactamase encoding gene from pLA230 through PCR method; obtain E.coli transformant carrying pTA2 with insert gene β-lactamase from pLA230 facilitated by TA cloning method; and obtain information related to the source, structure, and group of β-lactamase protein obtained through sequencing data analysis using bioinformatics software in silico. This study succeeded in obtaining 861 bp of β-lactamase enzyme encoding gene from pLA230, which was confirmed based on the alignment data between the origin sequence of the recombinant plasmid map pLA230 with the clone sequence read by sequencing method. The 861 bp of β-lactamase coding gene amplified using TA cloning method in the pTA2 vector via heat shock transformation. Positive E. coli transformants were screened using blue-white screening that can grow well on selective media containing 100 mg/mL penicillin. Analysis using Blastp software showed that the gene most likely came from Klebsiella pneumoniae, Escherichia coli, or Acinetobacter haemolyticus with a percentage confidence level of 99.95%, 99.30% and 99.30%. Tertiary protein modeling using I-TASSER software showed 5 protein models with the highest C score of -0.07 in the 1st model. (c) The analysis results inform that the β-lactamase protein is a group of Extended Spectrum Beta Lactamase (ESBL) of class A which is a class of Serine Beta Lactamase (SBLs) with the greatest specific similarity to the SHV-1 β-lactamase type protein (Sulphydryl Variable beta lactamase) which is a type of β-lactamase enzyme that has specific activity on penicillin antibiotics, and TEM-1 β-lactamase protein (Analysis of Temoniera beta lactamase) which is a type of class A β-lactamase (SBLs) from Gram-positive and negative bacteria, with Enzyme Commission Number (EC) of 3. 2.5.6.

Copyright

© 2024 The Author(s)

Open Access

Open Access This chapter is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

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TY  - CONF
AU  - Meta Asha Oktaviana
AU  - Anggun Sari Anjarwati
AU  - Delia Wahyu Pangesti
AU  - Rizqi Layli Khusufi
AU  - Suharti
AU  - Norazlinaliza Salim
AU  - Norman Yoshi Haryono
PY  - 2024
DA  - 2024/06/21
TI  - Cloning of β-Lactamase Encoding Gene as the Initiation Approach in Providing High Quality Milk
BT  - Proceedings of the 4th International Conference on Halal Development (4th ICHaD 2023)
PB  - Atlantis Press
SP  - 101
EP  - 115
SN  - 2352-5398
UR  - https://doi.org/10.2991/978-2-38476-261-3_10
DO  - 10.2991/978-2-38476-261-3_10
ID  - Oktaviana2024
ER  -