I-Hielscher Ultrasound Technology

I-Ultrasonic Lysis ka-E. Coli

  • Ama-bacterium e-coli yi-bacteria asetshenziselwa kakhulu kwi-microbiology kanye ne-biotechnology.
  • Ama-ultrasonic cell disruptors aletha imiphumela enokwethenjelwa futhi ephindaphindiwe ye-lysis ye-E. coli.
  • Ukuvuthwa okunamandla nokulawulwa kahle kwe-cavitation ne-shear kubangela ukuphazanyiswa okuphelele nokukhiqizwa okuphezulu (isib. Amaprotheni, i-DNA).

Ukuphazamiseka Kwamaselula nge-Cavitation

Ama-bacteria e-Escherichia coli ahlolwe ngokuthembekile usebenzisa ama-homogenizers ama-ultrasonic.Ultrasonic probe-hlobo homogenizers isebenza nge-approx. Imijikelezo engu-20,000 ngomzuzwana (ngo-20kHz) futhi kubangele ukucwiliswa kweziphuzo noma uketshezi. Izindawo ze-Acoustic ezincane ze-cavitation ezinjengezingcindezi ezinjenge-vacuum kanye namazinga aphezulu okushisa aphulukisa amaseli. Nakuba amazinga okushisa angafinyelela ama-degrees Celsius ayizinkulungwane eziningana, amavolumu ama-cavitation amancane kakhulu awasho ukushisa kakhulu inqubo. I-ultrasound eyenziwe i-acoustic cavitation ne-shear forces iphoqa noma iphula isikhumba se-E.coli – kuye ngokuthi izilungiselelo zedivayisi ye-homogenizer ye-ultrasonic.

Izinzuzo ze-Ultrasonic Lysis

  • ukulawula okuqondile kwe-lysis (amandla, ubukhulu, izinga lokushisa)
  • I-adapttion evumelekile kuma-sampuli athile
  • ukushisa lokushisa
  • for amancane kakhulu kuya amasampula ezinkulu kakhulu (μL kuya amalitha)
  • imithi yokwelapha ehlanzekile
  • ukulingana okulinganiselwe kusuka ebhodini kuya ekukhiqizeni
Idivaysi ye-ultrasonic i-VialTweeter ivumela isampuli yokulungiselela okukodwa ngesikhathi esisodwa kuya ngaphansi kwezimo eziyizinqubo ezifanayo. (Chofoza ukuze ukhulise!)

I-VialTweeter for lysis ultrasonic

Isicelo solwazi




Qaphela kwethu Inqubomgomo yobumfihlo.


Yize i-lysis yamakhemikhali ne-enzymtic ingaba yinkinga – njengoba i-chemical lysis ingashintsha izakhiwo zamaphrotheni futhi ingenise izinkinga zokuhlanza kanye ne-enzymatic lysis kudinga isikhathi eside sokufakelwa ukuphuza futhi asikwenziwe kabusha – Ukuphazanyiswa kwe-ultrasonic kuyinkimbinkimbi, indlela esheshayo yokuphazanyiswa kwamaseli.
I-ultrasonic lysis isekelwe emandleni okusebenza kuphela. Awekho amakhemikhali afakiwe, i-sonication iphula udonga lwamaseli ngamandla emisindo. I-lysis ye-Chemical ingashintsha isakhiwo seprotheyini bese iveza izinkinga zokuhlanza. ukuphazanyiswa kwe-enzymatic kudinga isikhathi eside sokukhuphuka futhi asikwenziwe kabusha.

Izincomo ezijwayelekile

UP400St i-ultrasonicator nge-flow reactorI-Sonication iyindlela ewuthandwa kakhulu yokufaka lysing encane kakhulu, ephakathi nendawo enkulu yokumiswa kwamaseli – kusuka pico-amalitha kuze kube 100L / hr (usebenzisa ultrasonic flow cell). Amaseli ahlotshiswe yi-shear eyisikali kanye ne-cavitation. I-DNA nayo ikhiqizwa ngesikhathi i-sonication, ngakho-ke akudingekile ukwengeza i-DNase ekumisweni kweseli.
Ukulawula ukushisa:
Ngaphambi kokupholisa isampuli bese ugcina isampula ngenkathi u-sonication eqhwa, isampula ukuchithwa okushisayo kwesampula kungavinjelwa kalula.
Okufanelekile, amasampula kufanele agcinwe iqhwa elibandayo ngenkathi i-lysis, kodwa ikakhulukazi isampuli lanele ngokwanele uma izinga lokushisa lingakhuphuki ngaphezu kokushisa komkhiqizo noma umthombo wesisindo. Ngakho-ke kunconywa, ukugcina ukumiswa kweqhwa futhi ukukhulumisana ngama-ultrasonics ama-pulses amafushane ka-5-10 amasekhondi nokuma kwe-10-30 sec. Ngesikhathi sokumisa, ukushisa kungahlakazeka ukuze kuvuselelwe ukushisa okuphansi. Ukuze uthole amasampuli amangqamuzana weselula, amakhemikhali ahlukahlukene egeleza nge-cooling jackets ayatholakala.

Izivumelwano zokulungiselela ama-E. Coli Lysates

Ukuhlaziywa kwemifanekiso kanye nokuhlanzwa kwamaprotheni avuselelayo

I-pellet ye-E. coli yanikezwa nge-ultrasonic system UP100H (Hielscher). Ngalesi sizathu, i-pellet yeselula yaphinda iphinde isetshenziswe ku-lled buffer buffer (i-Tris-HCl engama-50mM pH = 7.5, i-NaCl eyi-100 mM, i-5mM DTT, i-1 mM PMSF) futhi ihlile eqhweni ngamaminithi angu-10. Khona-ke, ukumiswa kwamaseli kwanikezwa ukuqhuma okufushane okuyi-10 s okulandelwa ngu-30 s ukuphumula. Okokugcina, ama-debris ekhishwayo asuswe yi-ultracentrifugation e-4 ° C ngamaminithi angu-15 ku-14000 rpm. Ukuze uthole ukuqinisekiswa kwenkulumo ye-rPR, i-supernatant yayiqhutshwa ku-12% wegeri le-polyacrylamide futhi ihlaziywe yi-SDS-PAGE ne-Western blotting. Ukuhlanzwa kwe-rPR kwenziwa ngokusebenzisa i-Ni2+-NTA resin (i-Invitrogen, e-USA) ngokusho komhlahlandlela womkhiqizi. Kulesi sigaba, kwasetshenziswa indlela yokuhlanza. Ukuhlanzeka kweprotheyini ehlanziwe kwahlolwa ngokusebenzisa i-electrophoresis kwi-gel ye-12% ye-polyacrylamide kanye ne-Coomassie ekhombisa ubuluhlaza. Ukuhlanzwa kwamaprotheni okuhlanzwa kwakulinganiswa yi-micro BCA protein assay kit (PIERCE, USA). (Azarnezhad et al. 2016)

I-ultrasonic cell iphazamisa u-UP100H (100W) for lysis, ukuphazanyiswa kwamaseli kanye nokugqoka i-DNA.

I-homogenizer ye-ultrasonic UP100H (100W)

Ukukhula Kwamaselula, Ukuguquka nokulungiswa kwe-E. coli Cell Extracts

I-SeqA ne-RNA polymerase i-ChIP-Chip E. coli MG1655 noma i-MG1655 i-DaseAA yakhula ngo-37 ° C kuya ku-OD600 cishe ngo-0.15 ku-50 ml LB (+ 0.2% i-glucose) ngaphambi kuka-27 μl we-formaldehyde (37%) nge-ml ephakathi kwanezelwa (ukuhlushwa kokugcina 1%). Ukuguqulwa kwenzelwa ukuthungatha kancane (100 rpm) ekamelweni lokushisa ngamaminithi angu-20 kulandelwa ukucinywa nge 10 ml we-2.5 M glycine (ukuhlushwa kokugcina 0.5 M). Ukuhlolwa kokushisa ukushisa, u-E. coli MG1655 wanyuka ngo-65 ml we-LB medium ku-30 ​​° C kuya ku-OD600 cishe ngo-0.3. Ngemva kwalokho ama-30 ml amasiko adluliselwa esikhwameni sokufudumala esanda kushisa ngo-43 ° C kanti okuseleyo kugcinwe ku-30 ​​° C. Ukweqa nokucima kwakunjengokuchazwe ngenhla ngaphandle kokuthi amaseli agcinwe ku-30 ​​noma ku-43 ° C amahora angu-5 ngaphambi kokuthuthumela kancane kancane ekamelweni lokushisa. Amaseli aqoqwa yi-centrifugation futhi ahlanza kabili nge-TBS ebandayo (pH7.5). Emva kokuvuselela ku-1 ml i-lysis buffer (i-10mM Tris (i-pH 8.0), i-20% i-sucrose, i-NaCl engu-50mM, i-10mM EDTA, i-10 mg / ml lysozyme) futhi i-incubation ku-37 ° C ngamaminithi angu-30 alandelwe ukufakwa kwe-4 ml IP i-buffer, amaseli ahanjiswa ngeqhwa ngezikhathi ezingu-12 amasekhondi angu-30 no-30 amasekhondi angu-30 UP400St Processor ultrasonic (Hielscher Ultrasonics GmbH) enegunya elingu-100%. Ngemuva kwe-centrifugation ngamaminithi angu-10 ku-9000 g, ama-aliquotes angu-800 μ a-supernatant agcinwe ku--20 ° C. (Waldminghaus 2010)

Ukukhiqizwa ngokweqile nokuhlanzwa kwama-enzyme.

Ukukhiqizwa ngokweqile kwe-decahistidine (i-His10) -amaprotheni ahlangene, i-E. coli BL21 (DE3) yaguqulwa nge-pET19b eyakhayo. I-preculture yasebusuku yayivunwa yi-centrifugation, kanti i-1% yayisetshenziselwa ukungena emasikweni okukhuluma. Amaseli athatha i-PET19mgtB akhule ngo-22 ° C kuze kube yilapho isibalo se-optical at 600 nm (OD600) we-0.7. Isiko sidluliselwe ku-17 ° C futhi sikhishwe ngu-100 μM IPTG. Ngemuva kwehora lesi-16, isiko sasivunwa yi-centrifugation ku-7 500 × g ku-4 ° C. Amaseli aphinda abuyele emanzini amaminerali angama-50 aphikisiwe-phosphate (PBS) ene-0.3 M i-NaCl e-pH 7.4 futhi ephazanyiswa yi-ultrasonication nge-sonotrode ye-S2 micro-tip UP200St i-ultrasonicator (i-Hielscher, iTeltow, eJalimane) emjikelezweni we-0.5 kanye nobukhulu be-75%.
Ukukhiqizwa ngokweqile kwe-decahistidine-tagged GtfC kwaxoshwa ku-37 ° C ku-OD600 of 0.6 nge 100 μM IPTG. Amaseli aphinde aqhutshelwa amahora angu-4, avunwe, futhi ahlaziyeke njengoba kuchazwe ngenhla nge-MgtB.
Ama-extract cell angasetshenzisiwe ayengama-centrifuged ku-15,000 × g no-4 ° C ku-sediment ama-debris amangqamuzana. Ama-extracts acacisiwe ayelayishwa ku-1-ml ye-HisTrap FF yamakholomu angcolile asebenzisa uhlelo lwe-ÄKTAprime Plus (i-GE Healthcare). Ama-enzyme ahlanzwa ngokusho komthetho womkhiqizi we-e-gradient elution wamaphrotheni akhethwe ngamakhemikhali. Izixazululo ezinamaprotheni ezinama-Eluted zaphelelwa kabili ngamakhemikhali angu-1 000 ama-PMS angu-50, i-pH 7.4, ne-NaCl engu-0.3 M ngo-4 ° C. Ukuhlanzwa kwahlaziywa yi-12% SDS-PAGE. I-protein yamanzi yayinqunywe indlela yaseBradford esebenzisa i-Roti-Quant (Carl Roth GmbH, eKarlsruhe, eJalimane). (Rabausch et al. 2013)

Ukukhipha amaprotheni avela ku-E. coli amabhaktheriya
Iprotheni ye-bait yesithakazelo (kulokhu, i-MTV1 ye-Arabidopsis thaliana) ifanelwe kumathegi we-GST futhi iboniswe ku-BL21 Escherichia coli (E. coli) amaseli.
1. Thatha i-pellet eyodwa ye-GST-MTV1 ne-GST (ehambisana ne-50 ml isiko lesibhaktheriya) bese uphinde uvuselele ngamunye ku-2.5 mL isikhukhula seqhwa elibandayo.
Sebenzisa i-ultrasonicator UP100H (ifakwe i-MS3 microtip-sonotrode eminyanisweni encane (2-5mL)) ukuphazamisa amangqamuzana angama-bacterium kuze kube yilapho ephikisiwe, okukhonjiswa ngophawu lokunciphisa kanye ne-viscosity eyandisiwe. Lokhu kufanele kwenziwe eqhweni, futhi kunconywa ukuba uhambisane ngezikhathi ezithile (isb. U-10 sec sonicating olandelwa yi-10 sec eqenjini njalonjalo). Kumelwe kuthathwe ukunakekelwa ukuze kungabonisi ngokuphakama okukhulu. Uma ukuqhuma noma ukubunjwa kwe-white precipitate kuyatholakala, ukuqina kufanele kudambise.
3. Dlulisa isisombululo se-bacysia lysed ku-1.5 mL amashubhu amancane ama-microcentrifuge at 4 ° C, 16,000 xg ama-20 min.

Amaprotheni allicin-modified ku-E. coli

I-VialTweeter i-ultrasonicator ekhululekile ye-homogenization encaneUkunqunywa kwe-Sulfhydryl Okuqukethwe ngu-5,5'-Dithiobis (2-nitrobenzoic acid) (DTNB) Assay
Umkhiqizo we-E. coli MG1655 ubusuku bonke wawusetshenziselwa ukungena emiphakathini ye-MOPS encane (1: 100). Isiko sakhula ngokweqile kuze kufinyelelwe i-A600 ka-0.4. Isiko sahlukaniswa sibe ngamasiko amathathu-15-ml wokuphathwa kwengcindezi. Isiko esingasetshenziswanga sasebenza njengesilawuli esingalungile. 0.79mM allicin (128 μg ml-1) noma i-diameter engu-1mM yanezelwa komunye wamasiko amabili asele ngalinye. Izimila zazifakwa emaminithini angu-15. I-5 ml yesiko ngasinye yayivunwa yi-centrifugation (8,525 × g, 4 ° C, 10 min). Amaseli ahlanzwa kabili nge-1 ml ye-PBS (i-NaCl 137 mM, i-2.7mM KCl, i-10 mM Na2I-HPO4, 2 mM KH2PO4, i-pH 7.4, igcinwe ngaphambi kokusetshenziswa) futhi ikhulu (13,000 × g, 4 ° C, 10 min). Amaseli aphinde aphinde abuyele e-lysis buffer (i-PBS ene-HMI ye-guanidinium engu-6m, i-pH 7.4) ngaphambi kokuphazanyiswa ngo-4 ° C nge-ultrasonication (I-VialTweeter ultrasonicator, Hielscher GmbH, eJalimane) (3 × 1 iminithi). Ama-debris e-cell aphethwe yi-centrifugation (13,000 × g, 4 ° C, 15 iminithi). I-supernatant yadluliselwa ku-3.5-ml ye-cupsette ye-QS-macro (10 mm) ene-bar yokugubha yamagnetic futhi ihlanganiswe ne-1 ml ye-lysis buffer. Ukuqothulwa kwamasampuli kwaqashwe ku-412 nm nge-Jasco V-650 i-spectrophotometer ehlonyelwe i-PSC-718 yokugcina izinga lokushisa (Jasco) ekamelweni lokushisa. I-100μl yesisombululo se-3mM i-dithiobis (2-nitrobenzoic acid) yanezelwa. Ukuqothulwa kwaqapha kwaze kwaba yilapho kufinyelelwa ukugcwala. Ukubalwa kwe-thiol yokucubungula kwenziwa nge-coefficient yokuqothula ε412 = 13,700 M-1 cm-1 for thio-2-nitrobenzoic acid (TNB). Ama-cellular concentrations ama-cellular ayalwa ngokususelwa kumthamo wama-E. coli amangqamuzana angu-6.7 × 10-15 ilitha kanye nesisindo se-A600 = 0.5 (okulingana no-1 × 108 amaseli ml-1 isiko). (UMüller et al. 2016)

Ukunqunywa kwe-Vivo Glutathione

I-E.coli MG1655 ikhulile ku-MOPS ephakathi okuphakathi kwenani elingu 200ml kuze ku-A600 ye-0.5 yafinyelelwa. Isiko sasihlukaniswa ngamasiko ama-50-ml ukuze ukwelashwa ngokucindezeleka. Ngemva kwamaminithi angu-15 okufakelwa ukufakelwa nge-allicin engama-0.79m, i-1 mM diamide, noma i-dimethyl sulfoxide (ukulawula), amaseli avuna ku-4,000g ku-4 ° C ngamaminithi angu-10. Amaseli ahlanjwe kabili nge-buffer ye-KPE ngaphambi kokuphindaphinda kwamapelisi ku-700μl ye-buffer ye-KPE. Ngokwe-deprotination, i-300l ye-10% (w / v) i-sulfosalicylic acid yanezelwa ngaphambi kokuphazamiseka kwamaseli nge-ultrasonication (3 x 1 iminithi; I-VialTweeter i-ultrasonicator). Ama-supernatants aqoqwe emva kokuqala kwe-centrifugation (30 min, 13,000g, 4 ° C). I-Sulfosalicylic acid concentrations yancipha ibe yi-1% ngokungezwa kwezinguquko ezingu-3 ze-KPE. Ukulinganisa kwe-glutathione ephelele kanye ne-GSSG kwenziwa njengoba kuchazwe ngenhla. Izibalo ze-glutathione zamangqamuzana zibalwa ngokususelwa kumthamo wama-E. coli amangqamuzana angu-6.7×10-15 ilitha kanye nesisindo se-A600 0.5 (okulingana no-1×108 amaseli ml-1 isiko). Amazinga we-GSH abalwa ngokukhipha i- 2 [GSSG] kusuka ku-glutathione esiphelele. (UMüller et al. 2016)

Ukuphazamisa i-ultrasonic yesilysis lysis kanye nesisetshenziswa sempahla yezinto eziphilayo (Chofoza ukuze ukhulise!)

I-Probe-type ultrasonicator UP400St

Ukuveza kwe-Human mAspAT ku-E. coli

I-ultrasonic cell iphazamisa u-UP400St (400W) ngokukhishwa kwendaba ye-intracellular (isib. Amaprotheni, i-organelles, i-DNA, i- RNA njll)Ikholomu eyodwa ye-E. coli BL21 (DE3) ephethe i-vector inkulumo engu-30 mL ye-Luria-Bertani (LB) ephakathi equkethe i-100μg / mL ampicillin, bese ihlwanyiswa ku-37ºC kuze kube yi-optical (OD)600) kufinyelelwe ku-0.6. Amaseli avuna nge-centrifugation ku-4,000 × g ngamaminithi angu-10, futhi abuye aphinde aphakanyiswe ku-3L isisindo esisha se-LB equkethe i-100μg / mL ampicillin.
Ngemuva kwalokho, inkulumo yeprotheni yenziwa nge-1 mM i-isopropyl β-∅-1-thiogalactopyranoside (i-IPTG) amahora angu-20 kuya ku-16ºC. Amaseli avunwa yi-centrifugation ku-8,000 × g ngamaminithi angu-15 futhi ahlanzwa nge-buffer A (i-NaM2, i-NaMI engu-20 MM, i-NaCl engu-0.5 M, pH 7.4). Amaseli angama-45g (isisindo samanzi) atholakala kumkhakha we-3 L. Ngemuva kwe-centrifugation, i-pellets yamaseli yabuyiselwa emuva ku-40 mL (ngomkhakha we-1 L) i-tampu ye-extraction ye-ice-abandayo, futhi ihlolwe yi-ultrasonication ngesikhathi sokushisa okubandayo kusetshenziswa i-an UP400St ithuluzi (uDkt. Hielscher GmbH, eJalimane). I-cell lysis yayineminyaka eyi-12 000 i-rpm imizuzu engu-15 ukuze ihlukaniswe izingxenyana ezinamanzi (supernatant) kanye ne-precipitated (pellet). (Jiang et al. 2015)

Ithebula elingezansi linikeza isibonakaliso somthamo wokucubungula we-ultrasonicators wethu:

Iqoqwana Ivolumu flow Rate Amadivayisi ezinconyiwe
0.5 kuya ku-1.5mL na I-VialTweeter
1 kuya ku-500mL 10 kuya ku-200mL / min UP100H
10 kuya ku-2000mL 20 kuya ku-400mL / min UP200Ht, UP400St
0.1 kuya ku-20L 0.2 kuya ku-4L / min I-UIP2000hdT
10 kuya ku-100L 2 kuya ku-10L / iminithi UIP4000
na 10 kuya ku-100L / iminithi UIP16000
na sikhulu yinhlanganisela UIP16000

Xhumana nathi! / Cela Us!

Sicela usebenzise ifomu elingezansi, uma ufisa ukucela ulwazi oluthe xaxa mayelana ne-homogenization yama-ultrasonic. Sizojabula ukukunikeza uhlelo lwe-ultrasonic ukuhlangabezana nezidingo zakho.









Sicela uqaphele wethu Inqubomgomo yobumfihlo.


Izincwadi / Izinkomba



Amaqiniso Okufanele Ukwazi

E.coli

I-Escherichia coli (E. coli) i-gram-negative, i-anaerobic, i-rod-shape, i-coliform bacterium ye-genus Escherichia evame ukutholakala emathunjini aphansi empilo efudumele (i-endotherms). Kunenqwaba yezinkinga ze-E. coli (noma ama-subtypes) anezici ezihlukahlukene. Iningi lezinkinga ze-E. coli azilimazi kubantu, isib. Izinkinga ze-B no-K-12 ezisetshenziselwa ukucwaninga kwezicelo ezenzelwe ukuhlolwa. Kodwa-ke, ezinye izinhlobo ziyingozi futhi zingabangela ukugula okukhulu.
U-E. coli udlala indima ebalulekile kwezobunjiniyela bezinto eziphilayo zanamuhla kanye ne-microbiology yezimboni ngoba amabhaktheriya kulula ukuwasebenzisa. Izicelo zamebhu ezivamile ezibandakanya ukusetshenziswa kwe-E. coli, isib. Ukudala i-deoxyribonucleic acid (i-DNA) ephindaphindiwe noma ukwenza izinto eziphilayo.
U-E. coli uyisiphephelo esihle kakhulu sokukhiqizwa kwamaprotheni we-heterologous, futhi izinhlelo eziningi zokukhulisa amaprotheni zitholakala ukukhiqiza amaprotheni abuyele e-E. coli. Ukusebenzisa i-plasmids evumela ukuphakama kwezinga eliphezulu lamaphrotheni, izakhi zofuzo zingangeniswa amabhaktheriya, okuvumela ukukhiqiza amaprotheni anjalo ngamanani aphezulu ezinkambisweni zokuvuna ezimbonini.
I-E.coli isetshenziselwa njengezimboni zamaseli ukukhiqiza i-insulin. Ezinye izicelo zihlanganisa ukusetshenziswa kwama-E. coli amangqamuzana ukuthuthukisa nokukhiqiza imishanguzo kanye nama-enzyme angenakulinganiswa, ukukhiqiza ama-biofuels, kanye nokuhlelwa kwemvelo.
Uhlobo lwe-K-12 luhlobo oluthile lwe-E. coli olwedlulele-olubonisa i-enzyme i-Alkaline Phosphatase (ALP). Lokhu kuguquka kwenzeka ngenxa yesici esakhiweni esihlale sinekhodi ye-enzyme. Uma izakhi zikhiqiza umkhiqizo ngaphandle kokuvimbela lokhu kuyaziwa ngokuthi umsebenzi we-constitutive. Leli fomu elithile lomuthambo lisetshenziselwa ukuhlukaniswa nokuhlanzwa kwe-ALP enzyme.

Ukukhishwa kwe-Ultrasonic DNA

Ama-ultrasonic shear amabutho yindlela evame ukusetshenziswa ukuhlukanisa neseli bese iphula i-DNA ibe yizicucu. I-cavitation ye-Acoustic iphula izindonga zezingqamuzana nezindwangu ukuze zikhishwe i-DNA yamaseli futhi zenze izingcezu ezingaba ngu-600 – I-800 bp ubude, ekulungele ukuhlaziywa.
Chofoza lapha ukuze ufunde kabanzi mayelana ne-ultrasonic homogenizers ye-DNA ukuhlukaniswa!