Method of regulating gene expression in a cell

Patent metadata

Field	Value
Publication number	US-11384358-B2
Application number	US-201816498465-A
Country	US
Kind code	B2
Filing date	Mar 29, 2018
Priority date	Mar 29, 2017
Publication date	Jul 12, 2022
Grant date	Jul 12, 2022

Abstract

The disclosure provides methods of making a tetracycline inducible expression system in a cell. The methods include providing the cell with a first nucleic acid sequence comprising a first promoter operably linked to a tetracycline repressor gene coding sequence, providing the cell with a second nucleic acid sequence comprising a second promoter operably linked to a coding sequence of a gene of interest wherein the second promoter is modified to include one or more tetracycline repressor protein binding sites, and determining the expression of the gene of interest in the presence or absence of tetracycline. The disclosure further provides nucleic acid sequences, vectors and cells including the tetracycline inducible modified promoter.

First claim

The invention claimed is:

1. A genetically engineered lactic acid bacterial cell comprising:
an agent-responsive element having a promoter and one or more transcription repressor protein binding sites,
wherein the agent-responsive element is operably linked to a target nucleic acid sequence,
wherein the agent-responsive element has at least 97% homology to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 10, 11, 12, 13, 14, and 15,
wherein a transcription repressor protein is bound to the one or more transcription repressor protein binding sites, and
wherein transcription of the target nucleic acid sequence into a corresponding mRNA is initiated upon binding of a cognate binding agent to the transcription repressor protein causing an allosteric or conformational change to the transcription repressor protein releasing the transcription repressor protein from the one or more transcription repressor protein binding sites.

2. The genetically engineered lactic acid bacterial cell of claim 1 , wherein the one or more transcriptional repressor protein binding sites is TetO, the transcriptional repressor protein is TetR, and the cognate binding agent is tetracycline.

3. The genetically engineered lactic acid bacterial cell of claim 1 , wherein the lactic acid bacterial cell is within the order Lactobacillales.

4. The genetically engineered lactic acid bacterial cell of claim 1 , wherein the lactic acid bacterial cell is selected from the group consisting of Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Carnobacterium, Enterococcus, Oenococcus, Tetragenococcus, Vagococcus , and Weisella.

5. The genetically engineered lactic acid bacterial cell of claim 1 , wherein the lactic acid bacterial cell is selected from the group consisting of Lactobacillus acetotolerans, Lactobacillus acidifarinae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarius, Lactobacillus alvei, Lactobacillus alvi, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylotrophicus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus animata, Lactobacillus antri, Lactobacillus apinorum, Lactobacillus apis, Lactobacillus apodemi, Lactobacillus aquaticus, Lactobacillus aviarius, Lactobacillus backii, Lactobacillus bifermentans, Lactobacillus bombi, Lactobacillus bombicola, Lactobacillus brantae, Lactobacillus brevis, Lactobacillus brevisimilis, Lactobacillus buchneri, Lactobacillus cacaonum, Lactobacillus camelliae, Lactobacillus capillatus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae, Lactobacillus catenefornis, Lactobacillus ceti, Lactobacillus coleohominis, Lactobacillus colini, Lactobacillus collinoides, Lactobacillus composti, Lactobacillus concavus, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curieae, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus equi, Lactobacillus equicursoris, Lactobacillus equigenerosi, Lactobacillus fabifermentans, Lactobacillus faecis, Lactobacillus faeni, Lactobacillus farciminis, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus floricola, Lactobacillus forum, Lactobacillus formosensis, Lactobacillus fornicalis, Lactobacillus fructivorans, Lactobacillus frumenti, Lactobacillus fuchuensis, Lactobacillus furfuricola, Lactobacillus futsaii, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus gastricus, Lactobacillus ghanensis, Lactobacillus gigeriorum, Lactobacillus ginsenosidimutans, Lactobacillus gorillae, Lactobacillus graminis, Lactobacillus guizhouensis, Lactobacillus halophilus, Lactobacillus hammesii, Lactobacillus hamsteri, Lactobacillus harbinensis, Lactobacillus hayakitensis, Lactobacillus heilongjiangensis, Lactobacillus helsingborgensis, Lactobacillus helveticus, Lactobacillus herbarum, Lactobacillus heterohiochii, Lactobacillus hilgardii, Lactobacillus hokkaidonensis, Lactobacillus hominis, Lactobacillus homohiochii, Lactobacillus hordei, Lactobacillus iatae, Lactobacillus iners, Lactobacillus ingluviei, Lactobacillus insectis, Lactobacillus insicii, Lactobacillus intermedius, Lactobacillus intestinalis, Lactobacillus iwatensis, Lactobacillus ixorae, Lactobacillus japonicus, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kalixensis, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus kimbladii, Lactobacillus kimchicus, Lactobacillus kimchiensis, Lactobacillus kisonensis, Lactobacillus kitasatonis, Lactobacillus koreensis, Lactobacillus kullabergensis, Lactobacillus kunkeei, Lactobacillus larvae, Lactobacillus leichmannii, Lactobacillus letivazi, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mellifer, Lactobacillus mellis, Lactobacillus melliventris, Lactobacillus micheneri, Lactobacillus mindensis, Lactobacillus mixtipabuli, Lactobacillus mobilis, Lactobacillus modestisalitolerans, Lactobacillus mucosae, Lactobacillus mudanjiangensis, Lactobacillus murinus, Lactobacillus nagelii, Lactobacillus namurensis, Lactobacillus nantensis, Lactobacillus nasuensis, Lactobacillus nenjiangensis, Lactobacillus nodensis, Lactobacillus odoratitofui, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus ori, Lactobacillus oryzae, Lactobacillus otakiensis, Lactobacillus ozensis, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus parabuchneri, Lactobacillus paracollinoides, Lactobacillus parafarraginis, Lactobacillus parakefiri, Lactobacillus paralimentarius, Lactobacillus paraplantarum, Lactobacillus pasteurii, Lactobacillus paucivorans, Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus plajomi, Lactobacillus plantarum, Lactobacillus pobuzihii, Lactobacillus pontis, Lactobacillus porcinae, Lactobacillus psittaci, Lactobacillus rapi, Lactobacillus rennanquilfy, Lactobacillus rennini, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rodentium, Lactobacillus rogosae, Lactobacillus rossiae, Lactobacillus ruminis, Lactobacillus saerimneri, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactobacillus saniviri, Lactobacillus satsumensis, Lactobacillus secaliphilus, Lactobacillus selangorensis, Lactobacillus senioris, Lactobacillus senmaizukei, Lactobacillus sharpeae, Lactobacillus shenzhenensis, Lactobacillus sicerae, Lactobacillus silagei, Lactobacillus siliginis, Lactobacillus similis, Lactobacillus songhuajiangensis, Lactobacillus spicheri, Lactobacillus sucicola, Lactobacillus suebicus, Lactobacillus sunkii, Lactobacillus taiwanensis, Lactobacillus thailandensis, Lactobacillus tucceti, Lactobacillus ultunensis, Lactobacillus uvarum, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus vermiforme, Lactobacillus vespulae, Lactobacillus vini, Lactobacillus wasatchensis, Lactobacillus xiangfangensis, Lactobacillus yonginensis , and Lactobacillus zymae.

6. The genetically engineered lactic acid bacterial cell of claim 1 , wherein the promoter sequence is the slpA promoter sequence.

7. The genetically engineered lactic acid bacterial cell of claim 1 , wherein the one or more transcription repressor proteins is a tetracycline repressor protein (TetR).

8. The genetically engineered lactic acid bacterial cell of claim 1 , wherein the one or more transcription repressor protein binding sites is a tetracycline repressor protein binding site.

9. The genetically engineered lactic acid bacterial cell of claim 8 , wherein the agent-responsive element has two tetracycline repressor protein binding sites between a transcriptional start site and a riboso

Assignees

• Harvard College

Inventors

• Debnath Anik
• Juarez Javier Fernandez
• Lee Henry Hung-Yi
• Church George M

Classifications

• C12N1/205

  Bacterial isolates · CPC title

• A61P29/00 Primary

  Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] · CPC title

• C12R2001/23

  Lactobacillus acidophilus · CPC title

• C12R2001/245

  Lactobacillus casei · CPC title

• C12N15/635 Primary

  Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline · CPC title