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The construction and purification of truncated N terminal and C terminal versions of LeuI, the Immunity gene of Leucocin A, from Leuconostoc gelidum 187-22
Thesis (MSc)--Stellenbosch University, 2025.
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Stellenbosch : Stellenbosch University
2025
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| _version_ | 1867613805234618368 |
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| access_status_str | Open Access |
| author | Tambe, Azraa |
| author2 | Beukes, Mervyn |
| author_browse | Beukes, Mervyn Tambe, Azraa |
| author_facet | Beukes, Mervyn Tambe, Azraa |
| author_sort | Tambe, Azraa |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2025. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/132412 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:41:58.332Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/132412 The construction and purification of truncated N terminal and C terminal versions of LeuI, the Immunity gene of Leucocin A, from Leuconostoc gelidum 187-22 Tambe, Azraa Beukes, Mervyn Stellenbosch University. Faculty of Science. Dept. of Biochemistry. Multidrug resistance Antibiotics Bacteriocins Drug resistance in microorganisms Proteins -- Genetics Leuconostoc UCTD Thesis (MSc)--Stellenbosch University, 2025. Tambe, A. 2025. The construction and purification of truncated N terminal and C terminal versions of LeuI, the Immunity gene of Leucocin A, from Leuconostoc gelidum 187-22. Unpublished masters thesis. Stellenbosch: Stellenbosch University [online]. Available: https://scholar.sun.ac.za/items/306ebc78-d89b-4bec-abbf-7da728425807 ENGLISH ABSTRACT: The discovery of antibiotics is one of the major achievements in the medical and scientific industries which helped to prevent the spread of bacterial infections. However, due to the overuse and misuse of antibiotics, many bacterial species have become resistant to antibiotics, with some pathogens becoming multi-drug-resistant. The rise of multidrug resistant pathogens reduced the effects of the frequently used antibiotics. Therefore, there is a need for the development of new antimicrobial agents that can be used to target these multi-drug-resistant pathogens. This led to the discovery and identification of bacteriocins. Bacteriocins are ribosomally synthesised peptides which are produced naturally by bacteria, with the production of these peptides generally occurring during a nutrient or resource-limited environment. Bacteriocins are effective against closely related strains, but it has been shown to have an effect against distantly related bacterial species. Bacteria that produce bacteriocins are immune to its effect due to the presence of a immunity gene which translates into a dedicated immunity protein. The immunity protein is responsible for producer self-protection, however the mode of action of the immunity protein from Leucocin A is poorly understood. To understand the mode of action of the immunity protein, Wild-type, truncated N and C-terminal versions of the immunity gene, LeuI, of Leucocin A produced from Leuconostoc gelidum 187-22 was investigated. The DNA from L. gelidium was isolated, and the full Immunity protein (F-LeuI) and the truncated N (NT_LeuI) and C (CT_LeuI) terminal versions of the immunity gene (LeuI) were successfully amplified using specifically designed PCR primers. The amplified fragments were cloned into the pET28a expression vector, and subsequently transformed into E. coli BL21 cells. Putative clones were confirmed by PCR, by having the expected band sizes (NT_LeuI = 129 bp, CT_LeuI = 228 bp and F_LeuI = 339). Positive clones were sequenced and were confirmed to be in the correct position. The recombinant peptides, pNT_129, pCT_228 and pFI_339, were expressed and purified via IMAC. The expressed purified peptide, F_LeuI was confirmed by SDS-PAGE and whereas a dot blot assay confirmed the expression of CT_LeuI. UPLC-MS confirmed the peptide mass of NT_LeuI; the truncated N-terminal immunity peptide was confirmed to be doubly charged with a mass of 1216.0280 Da. The monoisotopic mass was determined to be 2430.0259 Da which surprisingly did not correspond to theoretical mass expectations of 4309.98 Da. Bioinformatic protein-protein characterisation using Alpha fold Server predicted that both the NT_LeuI and CT_LeuI fragments interacted with the membrane bound LeuA-IICD manPTS complex and potentially play a role in providing self-immunity against Leu A. The alpha helices of LeuI are required for binding to the complex, with the flexible C-terminal tail recognising the C-terminal part of Leu A. AFRIKAANSE OPSOMMING: Die ontdekking van antibiotika is een van die groot prestasies in die mediese en wetenskaplike industrieë wat gehelp het om die verspreiding van bakteriële infeksies te voorkom. As gevolg van die oorbenutting en misbruik van antibiotika het baie bakteriese spesies egter weerstandbiedend teen antibiotika geword, met sommige patogene wat multi-middelweerstandig geword het. Die opkoms van multigeneesmiddelweerstandige patogene het die effekte van die gereeld gebruikte antibiotika verminder. Daarom is daar 'n behoefte aan die ontwikkeling van nuwe antimikrobiese middels wat gebruik kan word om hierdie multi-middel-weerstandige patogene te teiken. Dit het gelei tot die ontdekking en identifikasie van bakteriosiene. Bakteriosiene is ribosomaal gesintetiseerde peptiede wat natuurlik deur bakterieë geproduseer word, met die produksie van hierdie peptiede wat gewoonlik tydens 'n voedingstof- of hulpbronbeperkte omgewing plaasvind. Bakteriosiene is effektief teen naverwante stamme, maar daar is getoon dat dit 'n effek teen ver- verwante bakteriese spesies het. Bakterieë wat bakteriosiene produseer, is immuun teen die effek daarvan as gevolg van die teenwoordigheid van 'n immuniteitsgeen wat vertaal word in 'n toegewyde immuniteitsproteïen. Die immuniteitsproteïen is verantwoordelik vir produsent selfbeskerming, maar die werkingswyse van die immuniteitsproteïen van Leukosien A word swak verstaan. Om die werkingswyse van die immuniteitsproteïen te verstaan, is Wild-tipe, afgeknotte N en C-terminale weergawes van die immuniteitsgeen, LeuI, van Leucocin A geproduseer vanaf Leuconostoc gelidum 187-22 ondersoek. Die DNA van L. gelidium is geïsoleer en volle Immuniteitsproteïen(F-LeuI) en afgeknotte N (NT_LeuI) en C (CT_LeuI) terminale weergawes van die immuniteitsgeen (LeuI) is suksesvol geamplifiseer deur gebruik te maak van spesifiek ontwerpte PCR-inleiders. Die geamplifiseerde fragmente is in die pET28a-uitdrukkingsvektor gekloneer, en daarna in E. coli BL21-selle getransformeer. Vermeende klone is bevestig deur PCR, deur die verwagte bandgroottes te hê (NT_LeuI = 129 bp, CT_LeuI = 228 bp en F_LeuI = 339). Positiewe klone is gevolgorde en is bevestig om in die korrekte posisie te wees. Die rekombinante plasmiede, pNT_129, pCT_228 en pFI_339, is uitgedruk en gesuiwer via IMAC. Die uitgedrukte gesuiwerde peptied, F_LeuI is bevestig deur SDS-PAGE en terwyl 'n dot klad-toets die uitdrukking van CT_LeuI bevestig het. UPLC-MS het die peptiedmassa van NT_LeuI bevestig; die afgeknotte N-terminale immuniteitspeptied is bevestig dat dit dubbel gelaai is met 'n massa van 1216.0280 Da. Die monoisotopiese massa is bepaal as 2430.0259 Da wat verbasend genoeg nie ooreenstem met teoretiese massaverwagtinge van 4309.98 Da nie. Bioinformatiese proteïen-proteïen karakterisering het voorspel dat beide die NT_LeuI en CT_LeuI fragmente in wisselwerking met die membraangebonde LeuA-IICD manPTS kompleks en beide terminale punte speel 'n rol in die verskaffing van selfimmuniteit teen Leucocin A. Die alfa-helikse van LeuI word benodig vir binding aan die kompleks, met die buigsame C-terminale stert wat die C-terminale deel van Leucocin A herken. Masters 2025-06-06T08:17:06Z 2025-06-06T08:17:06Z 2025-03 Thesis https://scholar.sun.ac.za/handle/10019.1/132412 en Stellenbosch University viii, 79 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Multidrug resistance Antibiotics Bacteriocins Drug resistance in microorganisms Proteins -- Genetics Leuconostoc UCTD Tambe, Azraa The construction and purification of truncated N terminal and C terminal versions of LeuI, the Immunity gene of Leucocin A, from Leuconostoc gelidum 187-22 |
| title | The construction and purification of truncated N terminal and C terminal versions of LeuI, the Immunity gene of Leucocin A, from Leuconostoc gelidum 187-22 |
| title_full | The construction and purification of truncated N terminal and C terminal versions of LeuI, the Immunity gene of Leucocin A, from Leuconostoc gelidum 187-22 |
| title_fullStr | The construction and purification of truncated N terminal and C terminal versions of LeuI, the Immunity gene of Leucocin A, from Leuconostoc gelidum 187-22 |
| title_full_unstemmed | The construction and purification of truncated N terminal and C terminal versions of LeuI, the Immunity gene of Leucocin A, from Leuconostoc gelidum 187-22 |
| title_short | The construction and purification of truncated N terminal and C terminal versions of LeuI, the Immunity gene of Leucocin A, from Leuconostoc gelidum 187-22 |
| title_sort | construction and purification of truncated n terminal and c terminal versions of leui the immunity gene of leucocin a from leuconostoc gelidum 187 22 |
| topic | Multidrug resistance Antibiotics Bacteriocins Drug resistance in microorganisms Proteins -- Genetics Leuconostoc UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/132412 |
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