Ma `lumot

Luria Broth va Lysogeny Broth o'rtasida farq bormi?


Luriya Bertani va Luriya Broth o'rtasida farq bormi? Yoki ikkalasi ham bir xilmi? LB vositasi tayyor bo'lgandan keyin uni avtoklav qilish kerakmi?


Garchi ikkala ism ham juda keng tarqalgan bo'lsa -da, ikkalasi ham noto'g'ri. 1951 yilgi asl qog'ozda Bertani lizogenezni o'rgangan E.coli, shuning uchun u o'z ommaviy axborot vositalarini shu maqsadda "Lizogenli bulon" yoki qisqacha LB deb atadi. Keyingi o'n yilliklarda bu nom "Luriya Bertani" yoki "Luriya bulyoni" ga aylantirildi, bu noto'g'ri. Qo'shimcha ma'lumot olish uchun 1 va 2 havolalarga qarang.

Ikkinchi savol uchun: atrof muhitda mavjud bo'lgan mikroorganizmlarning ifloslanishi va ko'payishining oldini olish uchun tayyorgarlikdan so'ng, vositalarni avtoklavlash kerak. Faqat ommaviy axborot vositalarini steril qilish, biz aniqlangan mikroorganizmlar bilan ishlashimiz mumkinligiga ishonch hosil qiladi.

Manbalar:

  1. Lizogenez I. bo'yicha tadqiqotlar.
  2. LB Medium cheklovlari

LB muhiti nima uchun ishlatiladi?

To'liq javobni o'qish uchun bosing. Shu munosabat bilan nima uchun biz LB agardan foydalanamiz?

Luriya bulyoni (FUNT) hisoblanadi odatda ozuqa moddalariga boy vosita ishlatilgan laboratoriyada bakteriyalarni etishtirish. ning qo'shilishi agar ga FUNT natijada bakteriyalar keluvchi jel hosil bo'ladi mumkin ular kabi o'sadi bor hazm qila olmaydi agar lekin mumkin dan ovqatlanishni yig'ish FUNT ichida.

Ikkinchidan, LB agar nimadan yasalgan? FUNT Bulyon, shuningdek, FUNT o'rta, Lizogen bulyoni, Luriya bulyoni yoki Luria-Bertani muhiti bakteriyalarni etishtirish uchun ko'p ishlatiladigan ozuqaviy boy muhitdir. Birinchi marta 1951 yilda Juzeppe Bertani tomonidan tasvirlangan 1 litrli muhit 10 gramm tripton, 5 gramm xamirturush ekstrakti va 10 gramm natriy xloriddan iborat.

Shu nuqtai nazardan, LB agar va LB bulonidan qanday farq bor?

Agar murakkab jelatinli uglevod bo'lib, unga qo'shiladi LB bulyon, mikroblar madaniyati sifatida bakteriyalar o'sishi uchun jel hosil qilish uchun. FUNT pepton, xamirturush ekstrakti, NaCl va o'z ichiga olgan boy muhit hisoblanadi agar (qattiq muhit uchun).


LB Media

LB rekombinant etishtirish uchun ishlatiladigan eng keng tarqalgan vosita hisoblanadi Escherichia coli (E. coli). LB media Juzeppe Bertani tomonidan lizogeniya bo'yicha olib borgan tadqiqotlari tufayli "lizogeniya bulyoni" deb nomlangan. LB odatda noto'g'ri Luria-Bertani media, Luria Broth yoki Lennox Broth deb nomlanadi. LB muhiti boshqa turli xil fakultativ organizmlarni etishtirish uchun ham qo'llaniladi.

LB Media komponentlari

LB tez -tez ishlatilishining bir sababi shundaki, uni tayyorlash juda oson, chunki uning tarkibida bir necha ingredientlar, tripton, xamirturush ekstrakti va natriy xlor (NaCl) bor. Tripton, oshqozon osti bezi fermenti tripsin bilan kazeinni hazm qilish natijasida hosil bo'lgan peptidlar aralashmasi, azot va uglerod beradi. Xamirturush ekstrakti vitaminlar (shu jumladan B vitaminlari) va ba'zi iz elementlarini beradi. NaCl transport va osmotik muvozanat uchun natriy ionlarini beradi. E. coli ota-ona shtammlaridan biri bo'lgan K-12 shtammidan olingan E. coli Hozirgi kunda molekulyar biologiyada qo'llaniladigan B vitamini ishlab chiqarish etishmayapti.

LB ning tarixiy ma'lumotlari

LB - 1950 -yillardan beri Enterobacteriaceae -ni o'stirish va bakteriofag plastinkalarini aniqlash uchun ishlatilgan, oziqlanishga boy muhit. LB ko'plab turlar uchun yaxshi o'sishi bilan tez o'sishiga imkon beradi. 1951 yilda Juzeppe Bertani blyashka shakllanishini optimallashtirish uchun LB ni ishlab chiqdi Shigella Enterobacteriaceae ning indikator shtammi. Bugungi kunda LB media rekombinant shtammlarning o'sishi uchun eng keng tarqalgan vosita hisoblanadi E. coli. LB ning uchta umumiy formulasi mavjud: LB Miller, LB Lennox va LB Luria. LB Miller formulasi LB ning umumiy yoki standart formulasi bo'lsa -da, ba'zida hamma LB deb ataladi. LB ning muqobil formulalari NaCl kontsentratsiyasida farq qiladi (1-jadval).

Jadval 1. LB formulalari.

Tarkibi % Luriya (g/l) Lennoks (g/l) Miller (g/l)
Tripton 1.0% 10 10 10
Xamirturush ekstrakti 0.5% 5 5 5
Natriy xlorid (NaCl) 0,05, 0,5 yoki 1,0% 0.5 5 10

LB nomi bilan bog'liq chalkashliklar Bertani, Luriya va Lennoks tarixini hisobga olgan holda tushunarli. Juzeppe Bertani LB mediasini yaratganida Indiana universitetining Luriya laboratoriyasi a'zosi bo'lgan. Ed Lennox, shuningdek, Luriya laboratoriyasining a'zosi bo'lgan va Bertani bilan dastlabki lizogenez tajribalaridan foydalangan. Shigella. Salvador Luria 1955 yilda nashr etilgan maqolada u Bertanining asl formulasini ko'chirgan va LB ba'zan uning ilmiy darajasi tufayli Luriyaga noto'g'ri munosabatda bo'lgan va shuning uchun ham noto'g'ri Luria bulyon deb atalishi mumkin. Asl Bertani formulasi 1,0% Bakto tripton (10,0 g/L), 0,5% xamirturush ekstrakti (5,0 g/L), 1,0% NaCl (10,0 g/L), 0,1% glyukoza (1,0 g/L) pH 7,0 ga o'rnatildi. 1 N NaOH bilan. Avtoklavlashdan keyin glyukoza qo'shiladi. Vaqt o'tishi bilan glyukoza qo'shilishi LB ning barcha formulalaridan tushib ketdi. Miller nomi kitobdagi formuladan kelib chiqqan Molekulyar biologiya bo'yicha tajribalar Jefferi Miller tomonidan 1972 yilda nashr etilgan, unda glyukoza yo'q. LB ning asl va eng keng tarqalgan formulasi Miller 1,0% NaCl o'z ichiga oladi. 1955 yilda Ed Lennoks DNK sintezi mexanizmlarini o'rgangan E. coli osmotik stressga sezgir LB formulasini ishlab chiqdi, uning tarkibida tuzning yarmi, ya'ni 0,5% NaCl bor. Ushbu formulani LB Lennox deb atashadi. Bugungi kunda LB Lennox etishtirish uchun ishlatiladi E. coli tuzga sezgir antibiotiklardan foydalanganda, masalan, Blastasidin, Puromitsin va Zeotsin. Eng kam tuzni o'z ichiga olgan LB ning uchinchi formulasi LB Luriya deb ataladi. Ushbu formulada 0,05% NaCl mavjud. LB Luria kabi dengiz organizmlarini ajratish uchun ishlatiladi Vibrio xoleralari.

LBda o'sish mexanizmlari

LB media dastlab past zichlikdagi bakteriyalarni ko'paytirish uchun mo'ljallangan. Eksponensial o'sish, barqaror o'sish davri, OD tugashi bilan taxmin qilinadi600 (optik zichlik 600 nm) 0,6 dan 1,0 gacha. Ma'lumki, o'sishi E. coli LBda odatda OD bo'lganda to'xtaydi600 normal o'sish sharoitida taxminan 2,0 ga etadi, taxminan 0,6 mg ga to'g'ri keladi E. coli (quruq vazn) mL uchun. 2007 yilda D'Ari va uning hamkasblari LBning o'sish xususiyatlarini, xususan, fiziologiyasini o'rganib chiqishdi. E. coli K-12, bugungi kunda molekulyar biologiyada eng ko'p ishlatiladigan shtammlardan biri. Ular K-12 hujayralari LB Millerda yakuniy OD bilan o'stirilganligini ko'rsatdilar600 0,6-1,0 har doim ham bir xil fiziologik holatda emas. D'Ari va uning hamkasblari LBda dioksik o'sish (ikki fazali o'sish) haqida ilgari kuzatilgan kuzatuvlarni tasdiqladilar va eksponensial o'sish to'xtaganini qayd etdilar.

OD600 0,3, odatda taxmin qilinganidan ancha oldin. Ma'lumki E. coli Ma'lumki, pH darajasi 9.0 dan oshganda o'sishning yomonligi kuzatiladi va LB muhitining pH darajasi 9.0 ga yaqin o'zgarishi kam uchraydi. Biroq, D'Ari va hamkasblari LB pH qiymatini sozlaganlarida, o'sish egri chizig'iga hech qanday ta'sir ko'rsatmadi, holbuki ommaviy axborot vositalariga glyukoza qo'shilganda, madaniyatlar ODgacha o'sishi mumkin edi.600 6.49. Bu o'sish ekanligini ko'rsatadi E. coli LBda uglerod cheklangan. Ta'kidlanganidek, Bertanining LB ning asl formulasida 0,1% glyukoza mavjud edi. Ushbu tadqiqot shuni ko'rsatadiki, LB muntazam o'sish uchun yaxshi vosita bo'lsa -da, uni qayta ishlab chiqarish va eksponensial o'sishning uzoq davom etishi zarur bo'lgan fiziologik tadqiqotlar uchun ishlatmaslik kerak.

LB va tanlov

LB muhitiga organizmlar populyatsiyasini aniqlash yoki tanlash uchun qo'shilishi mumkin bo'lgan bir qator qo'shimchalar mavjud. Bir yoki bir nechta antibiotiklarga qarshilik ko'rsatish uchun yaratilgan hujayralarni tanlash uchun antibiotiklar ko'pincha LB muhitiga qo'shiladi. X-Gal (5-bromo-4-kloro-3-indolil-b-D-galaktopiranozid) yoki Bluo-Gal (galogenlangan indolil-b-galaktozid) ketma-ketlikni kiritish uchun ko'k-oq skrining uchun LB muhitiga qo'shilishi mumkin. b-galaktosidaza genining a qismini o'z ichiga olgan plazmidda ko'p klonlash joyi (MCS). Lak promotor tomonidan boshqariladigan genlarning ekspressiyasini qo'zg'atish uchun muhitga metabolizatsiya qilinmaydigan laktoza analogi IPTG (izopropil-beta-D-tiogalaktopiranozid) qo'shiladi. Hech qanday ketma-ketlik kiritilmagan bakteriyalarda MCS koloniyasi ko'k rangga ega bo'ladi, chunki X-Gal yoki Bluo-Gal 5-bromo-4-xloro-indoksilni hosil qilish uchun b-galaktozidaza bilan bo'linadi. Plazmidlar uchun b-galaktozidaza funktsional bo'lmaydi va koloniyalar oq bo'lib qoladi.


Yigirmanchi asr o'rtalarida lizogenez: P1, P2 va boshqa eksperimental tizimlar

Tadqiqot bilan shug'ullanayotgan ko'pchiligimiz afzal ko'rgan materialga, yaxshi sinab ko'rilgan texnikalar to'plamiga, hal qilinmagan muammolarning doimiy ro'yxatiga, narsalarni ko'rib chiqish yoki bajarish usullariga egamiz, biz ularni bir xil laboratoriyadagi hamkasblarimiz va boshqalar bilan baham ko'ramiz. do'stlar, sobiq hamkorlar yoki raqobatchilar bo'lsin, bir xil mutaxassislik sohasi. Bularning barchasi fan tarixchisi Hans-J örg Rheinberger (76, 77) tomonidan kiritilgan va ishlatilgan ȁ tajriba tizimi ” kontseptsiyasini o'z ichiga oladi. Uning kontseptsiyasi ancha moslashuvchan va metaforalarga boy bo'lib, tanqidiy tarixiy tekshiruvdan ko'ra shaxsiy nuqtai nazardan kelib chiqadigan hozirgi hikoyaga osongina moslashtirilishi mumkin. Materiallar, texnika va nomenklaturani ehtiyotkorlik bilan cheklash turli laboratoriyalar va bir xil sohadagi olimlarning turli avlodlari o'rtasida konstruktiv o'zaro ta'sir o'tkazishga imkon beradi. Albatta, haddan tashqari amalga oshirilgan bu jarayon yangi sohalardagi o'zgarishlarni bo'g'adi va ba'zi kashfiyotlarni kechiktiradi. Qirqinchi yillarning boshlarida bakteriofagni replikatsiya va rekombinatsiya sirlariga olib boradigan qirollik yo'li sifatida o'rganishni qat'iy targ'ib qilgan Maks Delbr, bu yo'lda ishlaydigan ishchilar uchun umumiy materialdan (T-faglar) foydalanish zarurligi haqida juda ochiq gapirdi. Escherichia coli B) va aniq standartlashtirilgan texnikalar (1). Albatta, T-faglar, odatda, bakteriyalarning asirga olinmaydigan o'ta xavfli parazitlari bo'lib, shuning uchun bizga bakteriofaglar va ularning bakterial xo'jayinlari o'rtasidagi yanada soyali o'zaro ta'sirlar haqida ko'rsatma bera olmagan: infektsiya irsiyat bilan uchrashadigan lizogenez haqida.

Lizogen va lizin hosil qilish atamasi bakteriofaglar kashf etilgandan keyin juda erta qo'llanilgan va dastlab keng tavsifli, tanqidiy bo'lmagan ma'noda ishlatilgan. Boshqa tomondan, o'z -o'zidan (ya'ni, tashqaridan aniq infektsiya bo'lmaganida) bakteriofag ishlab chiqargan, lekin yaxshi o'sgan, lizisning aniq dalilisiz bo'lmagan bakterial kulturalar 1922 yilning boshida ajratilgan edi. bir necha yil, frankofoniya tibbiy tadqiqot jamiyatida juda qattiq. Kontseptsiyaning rivojlanishi yaxshi umumlashtirilgan (68) bo‘lsa-da, fan tarixi va falsafasi nuqtai nazaridan jiddiy qarashga loyiqdir. Bahslarning mazmunini ishtirokchilardan biri Pol Flu (42, shuningdek, 84-bandga qarang) aytadi. Pasteur institutining Eug ène Wollman (90, 91) o'sha paytda lizogenez tushunchalarining genetik ta'sirini ko'rgan kam sonli kishilardan biri edi. O'shanda Evropaning Lotin mamlakatlarida genetika juda kam rivojlangan edi, immunologiya tibbiyot muassasalarida kunning yulduzi edi. Bundan tashqari, bahs-munozaralar kamdan-kam hollarda aniq va umumiy ma'lumotga ega bo'lgan eksperimentning bir turiga qaratildi. O'ttizinchi yillarning boshlarida lizogenez ta'rifiga erishildi (26), u hozirgi kunda ham amalda, aniqki, zamonaviy molekulyar ta'sirlarsiz. Embriologiyada ishlatiladigan nemischa "Anlage" so'zini Bernet va Makki (27), biz hozir profilaktika deb atagan narsaga qo'llashgan. Anlage yoki propage kontseptsiyasi hech kim lizogen bakteriya ichida yuqumli fag zarrachalari borligini ko'rsatishda turli usullarni qo'llagan holda muvaffaqiyatga erishmaganiga asoslangan edi. Ikkinchi Jahon urushi bu ishning ko'p qismini to'xtatdi. Wollmans vafot etdi va gripp fashistlarning kontslagerlarida zo'rg'a tirik qoldi. Burnet ko'proq tibbiy yo'naltirilgan ishlarni oldi.

Men lizogenez bilan 1949 yilning boshida, Cold Spring Spring portida, Milislav Demerec laboratoriyasida ilmiy xodim sifatida uchrashganman. Men streptomitsinga bog'liq bo'lgan o'z-o'zidan va indüksiyon mutatsiyani o'rganardim E. coli B zo'riqishi. Men ilgari hech qachon bakteriyalar bilan ishlamaganman. Bir paytlar sohasi nibbordek ko'rinadigan koloniyaga hayron bo'ldim, men buni Evelyn Witkin va marhum Gus Doermannga ko'rsatdim, ular baxtiga mening laboratoriyalarim xonam yonida edi. Ular aniq tushuntirishni, fagning ifloslanishini taklif qilishdi, lekin ulardan biriga “ qo'shildi Lizogeniya … ” (aslida o'sha paytda ishlatilgan inglizcha so'z lizogenez yoki lizogenlik edi) degan narsa ham bor edi. Men ilgari lizogenez haqida eshitmagan edim. (garchi men Pol Buxnerning endosimbiozlar haqidagi [25] asarlari bilan tanishgan bo'lsam ham) va kutubxonada qo'shimcha ma'lumot olish uchun qazishni boshladim.

Taxminan bir vaqtning o'zida, menga noma'lum, ikkita muhim voqea sodir bo'ldi. Madisonda, Ester va Joshua Lederberg, K-12 shtammlari ustida ishlash jarayonida. E. coli, keyin faqat bakteriyalar�n tashqari Pnevmokokk—Ma'lumki, genetik material almashinuvi, ota-ona shtammi bilan aloqa qilganda kutilmaganda lizisga uchragan mutant ajratilgan. Ular K-12 asl shtammi ilgari hech qanday shubha bo'lmagan fag uchun lizogen xususiyatga ega degan xulosaga kelishdi va ular buni Tracy Sonnebornning kappa faktoriga o'xshash narsa deb o'ylab, lambda deb nomlashdi. Parametsiy. Bu haqda Witkinning norasmiy Microbial Genetics Bulletin nashrining birinchi sonida (1950 yil yanvar) qisqacha ma'lum qilingan (58). Parijda taniqli protozoolog va bakterial fiziolog André Lvoff fag zarralari lizogen bakteriyalar tomonidan qanday hosil bo'lishi haqidagi savolni oldi. Muammo katta miqdordagi individual yoki oz miqdordagi lizogen bakteriyalarning to'g'ridan -to'g'ri mikromanipulyatsiyasi bilan jasorat bilan hujumga uchradi. Bacillus megaterium, mikroskop ostida bulon tomchisida yetishtiriladi. Vaqti-vaqti bilan bakteriyalarni o'rab turgan suyuqlik olib tashlandi, yangi bulon bilan almashtirildi va keyin bakteriofag borligi tekshirildi. Shu bilan birga, tomchidagi bakteriyalar soni qayd etildi va tomchida lizing bo'lishi mumkin bo'lgan bakteriyalar tekshirildi. Kuchli natija shundaki, tomchi faglarni o'z ichiga olganida, ular bir yoki bir nechta bakteriyalarning to'satdan lizisini kutish mumkin bo'lgan ko'p miqdorda mavjud edi. Aksincha, bakteriyalar fag hosil qilmasdan bir necha marta o'sishi va bo'linishi mumkinligi ko'rsatildi. Biroq, fag ishlab chiqarishning hujayra lizisi bilan o'zaro bog'liqligini darhol ko'rsatish mumkin emas edi. Keyinchalik bu topilmaga oydinlik kiritildi B. megaterium hujayralar o'z -o'zidan va samarasiz lysing xususiyatiga ega bo'lib, ko'zga ko'rinadigan hujayra arvohini qoldirgan, fag ishlab chiqarish bilan bog'liq lizis juda tez bo'lgan va bakteriyalar jismlarining mikroskopda ko'rinadigan izlari qolmagan [69, 70].

1949 yilning kuzida men Bloomingtondagi Indiana universitetida Salvador Luriyaga qo'shildim. Ish rejalarini tuzayotganda men lizogenezni o'rganishni taklif qildim. Luriya bundan juda xursand bo'lmadi, chunki u T2 ning fagulent fagining rivojlanishi bilan bog'liq boshqa muammolarni o'ylab topgan edi (men bir necha oy ishlaganman). Balki —, men taxmin qilyapmanki — u o'zining tadqiqot granti taklifida lizogeniya haqida gapirmagan.  .  .  . U baribir juda hamjihat edi va men lizogenezni o'rganishim mumkin bo'lgan ba'zi shtammlarni olishga harakat qilishni va'da qildi. Aslida, 1950 yil yanvar oyida biz Lederbergdan K-12 bilan ishlatilishi mumkin bo'lgan indikator shtammlarini, shuningdek lizogen klassik Lissabon shtammini oldik. E. coli yigirmanchi yillarning boshlarida M. Lissabon va L. Karr tomonidan ajratilgan shtamm, Shigella indikator. Men zudlik bilan ikkala shtamm ustida ishlay boshladim, ularning madaniy xususiyatlarini ko'rib chiqdim va o'sish va blyashka shakllanishini optimallashtirishga harakat qildim. K-12 va lambdaning aniq afzalligi shundaki, fagni o'rganish bilan bakterial rekombinatsiyani birlashtirish mumkin edi, chunki Lederberglar ham qila boshladilar. Muqobil variantdan foydalanishni talab qildi Shigella, rasman patogen bo'lib, u laboratoriya xavfsizligi choralarini qo'llashdan ko'ra qattiqroq choralarni talab qiladi E. coli. Afsuski, bir necha hafta o'tgach, Luriya Joshua Lederberg bilan uchrashdi va agar men lambda ustida ishlamagan bo'lsam, u va Esterni afzal ko'rishini tushundi. O'sha paytda men juda norozi bo'ldim, garchi men Lederberglarning iltimosi ularning huquqlari doirasida ekanligini tan oldim, chunki ularning lambda kashfiyoti hali ochiq adabiyotda nashr etilmagan edi. Luriya, Jim Uotson (o'sha paytda Bloomingtonda aspiranturaning oxirgi kursida) va men Luriyaning ish stoli uchun burchak kesilgan kichik laboratoriya bilan bo'lishardik. Kech tushdan keyin biz Lederberglarning iltimosini inobatga olgan holda qanday davom etish haqida jiddiy muhokama qildik. Esimda, Jim o'zining baland ovozi bilan u odatda patogen bo'lgan laboratoriyada bo'lishni xohlamasligini aytdi. Shigella. Shunga qaramay, Luriya meni lambdani hech bo'lmaganda bir muddat yolg'iz qoldirishga ko'ndirdi va men ishlov berishda ehtiyot bo'lish masalasini qabul qildim. Shigella. Orqaga nazar tashlasak, bu kichik epizod menga hech qachon lambda bilan bog'liq holda organizm uchun to'g'ri his -tuyg'ularni rivojlantirishga imkon bermaganga o'xshaydi [43].

Lissabon shtammidan foydalanish (qisqartirish uchun Li sifatida tasvirlangan) va Shigella, Men asosan Lvoff bilan bir xil muammoni o'rganishga kirishdim. Dissertatsiya ishim davomida mikromanipulyatsiya charchoqlarini boshdan kechirganimdan so'ng (4), uning yondashuvidan foydalanish g'oyasi hech qachon xayolimdan chiqmagan. Bundan tashqari, lizogenli fag ishlab chiqarish B. megaterium boshqa lizogen shtammlarga (bir necha foiz erkin fag) nisbatan odatdagidan yuqori (masalan, har ikki bakteriya uchun bitta erkin faj zarrachasi), shuning uchun to'g'ridan -to'g'ri mikroskopik yondashuv ko'pchilik shtammlar bilan muvaffaqiyatli bo'lmaydi. Men birinchi bo'lib izolyatsiya qildim Shigella streptomitsinga chidamli mutant (keyinchalik nomi bilan tanilgan) Sh/s yoki Sh-16) va Li lizogen tomonidan ishlab chiqarilgan fag streptomitsinga ta'sir qilmaganligini ko'rsatdi. Keyin men “m modifikatsiyalangan bitta portlash tajribasini o'rnatdim, unda eksponentli ravishda o'sib borayotgan lizogen bakteriyalar (har qanday erkin fagni yo'q qilish uchun yuvilgan) naychalar to'plamiga taqsimlandi va inkubatsiyadan so'ng har bir naychaning butun tarkibi streptomitsinga chidamli indikator va bir tomchi streptomitsin bilan qoplangan. Bu usul plastinkada streptomitsin bo'lmaganda lizogen bakteriyalar ishlab chiqaradigan fagni emas, faqat qoplama vaqtida mavjud bo'lgan erkin fagni baholaydi. Agar fag lizogen o'sishi jarayonida doimiy ravishda ishlab chiqarilgan bo'lsa, plitalar tasodifiy taqsimlangan blyashkalarga ega bo'lar edi. Agar lizogen madaniyatlar faj sezadigan hujayraga virusli faj yuqtirilgandek, faj hosil qilsa, ko'p plastinkalarda blyashka bo'lmaydi va bir nechta blyashka ko'p bo'ladi, ya'ni, bitta bakteriyaning barcha fag avlodlari. Parametrlar sozlangandan so'ng, tajriba chiroyli ishladi (5). Lizogen tomonidan fag ishlab chiqarilishi uzluksiz ekanligini, kamdan -kam uchraydigan fag portlashlarini tasdiqladi. Shunday qilib, bir kunlik ish o'z -o'zidan fag ishlab chiqarish chastotasini, hatto juda past darajagacha (Li shtammining 45000 hujayra avlodiga bitta portlash) va o'rtacha portlash hajmini o'lchash imkonini berdi: mikromanipulyatsiya oylarida bittadan ko'pini olish mumkin edi. Ammo ajablanib bo'ldi: Li shtammi kulturalaridan olingan fag blyashka hajmi bo'yicha juda xilma-xil ekanligi ma'lum bo'lsa-da, mening tajribamda blyashka turli xil portlashlarda boshqacha ko'rinardi. Keyingi tadqiqotlar shuni ko'rsatdiki, Li shtammi haqiqatan ham immunologik jihatdan bir-biridan farq qiluvchi uchta turdagi faglarni ishlab chiqargan, men ularni P1, P2 va P3 deb nomlaganman, ularning har uchasi ham mustaqil ravishda lizogeniyani o'rnatishga qodir. Shigella deformatsiya va qoida tariqasida ular bir-biridan mustaqil ravishda bir hil portlashlarda ishlab chiqarilgan (5). Men Li shtammidagi faglar bilan ishlashni davom ettirdim, bunda P2 ga alohida e'tibor qaratdim, bunda turli blyashka tipidagi mutantlarni tanib olish nisbatan oson edi.

Shu bilan birga, Pasterda Lvof va bir nechta hamkorlar lizogen hujayraning normal o'sishidan fag portlashining o'z joniga qasd qilishiga o'tish qaroriga ta'sir ko'rsatadigan sharoitlarni izlashdi. Turli xil kimyoviy muolajalar muvaffaqiyatsizlikka uchraganidan so'ng, ular ajoyib muvaffaqiyatga erishdilar: ultrabinafsha nurlarining juda kichik dozasi ta'sir qilish ularning lizogen tarkibidagi deyarli barcha bakteriyalarni keltirib chiqardi. B. megaterium lizisga olib keladi va fag portlashini hosil qiladi (71). Yangi hodisa deb atalgan ultrabinafsha induktsiyaning kashf etilishi lizogeniyaga katta e'tiborni tortdi. Hatto bir necha yil oldin bu borada shubha bildirgan Delbr va#x000fcck ham, lizogenezani o'z ichiga olgan, asosan, Jeneva universitetining fizika professori, hamkasbi Jan Vayglning ishtiyoqi bilan. O'shanda erta nafaqaga chiqdi va faglar bo'yicha tadqiqotga kirishdi (83). Ko'p o'tmay, faj ham borligi aniqlandi Pseudomonas (51) va lambda E. coli K-12 (89) ultrabinafsha nurlar ta'sirida paydo bo'lishi mumkin. Boshqa tomondan, mening urinishlarim Li yoki Sh(P2) (ya'ni, Shigella P2 uchun lizogenli bo'lgan izolatlar) umuman manfiy edi. Bu umidsizlikka uchradi, lekin ayni paytda lizogen tizimlar o'rtasida asosiy biologik farqlar bo'lishi mumkinligining birinchi belgisi edi.

Lizogeniyaga oid bir qancha qiziqarli savollar ochiq edi. O'rnatilgan lizogen bakteriyada bitta profag nusxasi yoki ko'p nusxasi bo'ladimi? Agar bittasi bo'lsa, u qanday qilib hujayralar bo'linishida muntazam ravishda ajratiladi? Va superinfektsiyaga qarshi immunitet qanday edi? Immunitet o'ta yuqumli fagning rivojlanishiga to'sqinlik qiladigan, tarqaladigan, profilaktikaga xos bo'lgan mahsulot (immunitet moddasi ”, “repressor ”) tufayli bo'lganmi yoki maxsus bakterial sayt bilan zarur o'zaro ta'sir natijasida bo'lganmi? ? Ester Lederberg tomonidan 1950 yilgi hisobotida tilga olingan dastlabki bakterial xochlar K-12 da lambda lizogeniyasi va ba'zi genetik belgilar o'rtasida murakkab bo'lsa-da, qandaydir bog'liqlik borligini ko'rsatdi. Sitoplazmatik elementlar populyatsiyasini xromosomal nazorat qilish imkoniyati (kappa holatida bo'lgani kabi) Parametsiy) chiqarib tashlanmagan. Men P2 blyashka tipidagi mutatsiyalarni superinfeksiyada marker sifatida ishlatardim Sh (P2) lizogen hujayralar, superinfektsiyalovchi fagning taqdirini kuzatishga harakat qiladilar. Ma'lum bo'lishicha, ikkinchisi lizogenning immunitet tizimi tomonidan buzilmagan yoki rad etilmagan, u faqat replikatsiyada bloklangan (superinfektsiyadan oldingi profilaktika sifatida) va lizogen o'sishda va bo'linishda davom etar ekan, qiz hujayralarga tasodifiy taqsimlangan. . Qachonki uni olib yurgan hujayra lizga uchraganda, superinfektsiyalovchi fag portlashda, asosan, profil bilan bir xilda ishtirok etadi. Kamdan -kam hollarda, doimiy profilaktika (yoki uning ba'zi genlari) o'rnini o'ta infektsion tip egallaydi. Kamdan -kam hollarda barqaror ikki baravar lizogenli shtamm o'rnatiladi. Lizogenlash qobiliyatini yo'qotgan P2 mutanti (aniq blyashka turi “weak virulent ”) bir necha hujayrali avlodlar uchun bloklangan holatda saqlanishi mumkin va kamdan -kam hollarda hatto o'zini ikkinchi profil kabi tutadi, ya'ni hamma meros bo'lib o'tadi. tug'ma hujayralar, agar dastlabki profilaktika (loyqa blyashka) mavjud bo'lsa, u immunitetga ta'sir qiluvchi retsessiv mutatsiyaga ega bo'lgan mutantga o'xshab o'zini tutardi. Bu natijalar (6, 7, 8, 9, 10) murakkabroq talqinlarga ochiq boʻlsa-da, har bir bakterial hujayra (aniqrogʻi, har bir nukleoid uchun) bitta profag yoki istisno tariqasida ikkita gʻoyani ham, oʻziga xos kontseptsiyani ham qoʻllab-quvvatladi. mahsulotni superinfektsiyalovchi fag bilan o'zaro ta'sir qiladi. Ayni paytda, yangi tur E. coli, keyinroq chaqirdi C (16) sahnada paydo bo'ldi, bu bilvosita Kavalli va Xeslot (35) tomonidan topilgan natijadir. C shtammi lambda va Li shtammining barcha faglariga sezgir edi, P2 bilan yaxshi blyashka berdi, bundan tashqari (35) K-12 shtammlari bilan kesishadi. Asta-sekin men o'zimdan o'tdim Shigella P2 bilan ishlashning ko'p qismi uchun C shtammi, keyin esa profagning xromosoma joylashuvini aniqlash uchun bakterial xochlar amalga oshirilishi mumkin (10, 14). Men bu erda P2 bilan ishlashni ta'kidlagan bo'lsam-da, ko'pchilik o'quvchilar o'sha vaqt ichida (1951 yildan 1957 yilgacha) K-12 va lambda genetikasini tushunishda tez o'sishga erishilganini bilishadi (53) uning ikkita asosiy muallifi tomonidan ko'rib chiqilgan. . Ma'lum bo'lishicha, lambda profagining o'zi, aslida, bakterial xromosomada mumkin bo'lgan yagona joyda aniqlangan. Bog'lanishning batafsil tahlillari (28) va o'sha paytdagi xromosomalar doirasi haqidagi yangi tushunchalar Allan Kempbellning taniqli integratsiya modelini taklifiga olib keldi.

Lizogenezdan tashqari, P1 va P2 ham bakterial genetikaning erta rivojlanishiga o'z hissasini qo'shdi. (P3 bilan hech qachon bajarilmagan.) Misol tariqasida “host-boshqariladigan variatsiya” bo'lishi mumkin, bu endi ko'proq ȁkrestriksiya va modifikatsiya deb ataladi”.” Men buni P2 da payqadim (cheklovchi xost sifatida B shtammidan foydalangan holda). , Shigella standart uy egasi bo'lish) va undan nima qilishni bilmasdim. Men tez-tez yozishib turadigan Jan Vaygl buni lambdada payqagan (ruxsat beruvchi xost sifatida C shtammidan foydalanilgan, K-12 standart xost hisoblanadi). Mening natijalarimdan xabardor bo'lib, u darhol ikkita topilmaning o'xshashligini tan oldi. Bundan biroz oldin, Luriya (66) aytganidek, kichik laboratoriya baxtsiz hodisasi, uy egasi tomonidan boshqariladigan o'zgaruvchanlik holati bo'lsa ham, boshqa kashfiyotga olib keldi (67). Garchi o'sha paytda hech qanday qoniqarli mexanik tushuntirish ko'rinmasa -da, biz va Jan ikkimiz mutlaqo mustaqil bo'lgan tizimlarimiz o'rtasidagi parallellikdan dalda oldik va topilmalarimizni birgalikda nashr etishga qaror qildik (16). Ikki xil tizimda, turli laboratoriyalarda kuzatilgan yangi hodisaning bir xil maqolada, qiyosiy tarzda tasvirlanishi kamdan-kam uchraydi. Bu dalillarni kuchaytirdi va hodisaning umumiyligiga ishora qildi, shu bilan birga fan va insoniy ishlarda raqobatga qarshi hamkorlik uchun ball to'pladi. (Shunga o'xshash voqea, bir necha yil o'tgach, Ren é Tomas va Elizabet Bertani [87] tomonidan yozilgan, u lambda va P2 bilan parallel ravishda o'tkazilgan tajribalar, immunitet repressorining ish uslubini aniqroq aniqlagan.) P2 dyuym E. coli B yana bir kutilmagan topilmaga olib keldi: bu an'anaviy fag xost shtammida nuqsonli profag (P2 bilan bog'liq, ammo immunitetning o'ziga xosligi boshqacha) mavjudligi (37). Bugungi kunda nuqsonli profilaktika deyarli har kuni bakteriyalarni genomik tahlilida topiladi.

P1, shuningdek, ba'zi kutilmagan hodisalar berdi. Uning lizogenezining paydo bo'lishi Shigella deyarli harorat bilan nazorat qilingan (17): lizogenlashning juda yuqori chastotalari va infektsiyalangan bakteriyalar infektsiyadan keyin 20 ଌ da saqlanganida, faj ishlab chiqarilmasligi, faj ishlab chiqarilishi 40 va#x000b0C da. Ko'rinib turibdiki, P1 kamida ikkita mutatsion bosqich orqali moslashtirilgan, chunki bu xususiyat yo'qolgan. k va v (60, 88), K-12 da yanada samarali o'sadi va hech kim asl yovvoyi turni o'rganishga qaytmadi. Shigella. Bunday e'tiborsizlikning sababi, albatta, P1 transduktsiyaga qodir ekanligi aniqlandi va deyarli faqat K-12 hosilalarida qo'llanila boshlandi. Transduktsiya 1951 yilda Zinder va Lederberg tomonidan kashf etilgan Salmonella (94) va P22 fagi bilan bog'liq bo'lib, bir-biri bilan chambarchas bog'langan mutatsiyalarni genetik tahlil qilishga imkon berdi, ammo yalpi xaritalash yo'q. Boshqa tomondan, nozik tuzilmani xaritalash hali ham amaliy bo'lmagan E. coli K-12. P1 ning ushbu qobiliyatini kashf etganligi uchun o'sha paytda biologiyaga o'tish jarayonida bo'lgan fizik Ed Lennoks sharafiga sazovor bo'ldi, u 1954 yil boshida bir kuni ertalab laboratoriyamizga kirib: “Jo, keling, faglaringiz transduktsiya qila oladimi yoki yo'qligini tekshirib ko'raylik!& #x0201d Menda bu fikrni sinab ko'rish uchun ishlatilishi mumkin bo'lgan C turidagi auxotrofik mutantlar bor edi. Ertasi kuni biz tajriba o'tkazdik va haqiqatan ham P1 (lekin P2 emas) juda samarali transduser ekanligi ma'lum bo'ldi. Menda hali ham Jan Vayglga 16-aprelda yozilgan maktubning nusxasi bor, u yerda men haddan tashqari ishtiyoq bilan shunday deb yozgan edim: 𠇋iz bir haftani katta hayajonda o'tkazdik (ya'ni, Ed Lennoks va men). Mening P1 fajim transversiya qila oladi. Shigada o'stirilgan P1 arginin xarakterini o'zgartirishi mumkin coli C, galaktoza belgisi coli C va streptomitsin belgisi coli B. Yetarli?𠉫ir xil organizmdagi transduksiya va genetik rekombinatsiyani haroratning lizogenizatsiyaga ta'sirini solishtirish va transduksiyaga P2 profagini P1 va boshqalar va boshqalar orqali o'tkazishda muvaffaqiyatga erishish mumkin bo'ladi.”. o'sha bahorda (59) genetik rekombinatsiya bo'yicha Oak -Ridj yig'ilishida xabar bergan va undan keyin Lennoxning juda keng qamrovli maqolasi (60). Lennox (60) va Jeykob (52) lambda profagining bakterial markerlar bilan birgalikda o'tkazuvchanligini ko'rsatdi. Keyinchalik, uch xil uchastkadagi P2 profillari P1 (31) yordamida kotransduktsiya qilingan va yo'naltirilgan.

Keksa genetiklar yuqtirgan va o'tayotganda, hozirgi vaqtda molekulyar biologiya usullari elektron mikroskopi va ultratsentrifugatsiyaning poshnalarida paydo bo'lgan. Bir necha o'n yillar davomida lambda eksperimental tizimga bo'lgan ishtiyoqi lambda lizogenezining paradigmasini yaratdi (47, 48, 75, 82). Hozirgi vaqtda u molekulyar darajaga qadar shunchalik yaxshi ma'lumki, u foydali tarzda silikoda (2) modellashtirilgan. P1, shuningdek, g'ayrioddiy xromosoma harakati (50) tufayli dastlab transduksiya vositasi sifatida va keyinchalik o'z-o'zidan (93) ko'plab izdoshlarni jalb qildi. Ba'zilar, shu jumladan men ham, P2 ustida ishlashni davom ettirdim, garchi ba'zida izolyatsiyani his qilgan bo'lsam va tashvishlansam, bu harakatni oqlash uchun uning lambdaning farqi etarlicha muhim emas edi. Bu, menimcha, bunday bo'lmagan (12).

P2 va lambda zarralari tuzilish jihatidan farq qiladi. Lambdadan farqli o'laroq, P2 DNK replikatsiyasi butun reproduktiv tsikli davomida aylana-aylananing odatiy modeliga mos keladi (54, 64, 73, 79). Buni ikkita hayratlanarli topilma taklif qildi: P2 DNK replikatsiyasining xost hujayra Rep funktsiyasi (32) va cis-tasir etuvchi fag oqsili (62), juda boshqacha, virulent fag uchun bo'lgani kabi, φX174. Enkapsidatsiya to'g'ridan -to'g'ri monomerik doiralardan sodir bo'ladi (74). Bakterial xromosomada lambdanikidan farqli o'laroq, P2-ga xos birikish joylari mavjud (3, 14, 33, 55). P2 aralash infektsiyalarda genetik rekombinatsiya juda past chastotada sodir bo'ladi (11, 15): rekombinatsiyani rag'batlantirish uchun UV nurlanish yordamida P2 genetik xaritasini (13, 61) olish kerak edi. P2 tartibga solish davri, liziz va lizogeniya, lambdanikiga qaraganda oddiyroq (78). P2 profilining integratsiyalashuvi mexanizmi, o'ziga xos sayt rekombinatsiyasi bo'lsa-da, o'ziga xos xususiyatlarga ega (20, 30, 46), qisman derepressiya paytida profilaktika bo'linishini blokirovka qilish uchun javobgardir. Boshqa tomondan, P2da hech qanday ixtisoslashtirilgan transduksiya holati ma'lum emas, uning qo'shimcha hodisasi — bakterial markerni tushirilishi tasvirlangan [56]. P2 tadqiqotlari uchun katta turtki 1966 yilda Erix Six (81) tomonidan P4 ajoyib sun'iy yo'ldosh fagining kashf qilinishi va uning P2 va P2 kabi faglar bilan murakkab o'zaro ta'siri (“transactivation ”) edi (22, 29, 36, 65). . P2 ga xos bo'lgan juda qiziq kuzatuvlar hali to'liq tushunilmagan va qo'shimcha tadqiqotga loyiq: kichik molekulyar mahsulotga lizogenizatsiya (18, 19) hujayra sezuvchanligi (21) bo'yicha metabolik ta'sirlar (21) P2 nosessional gen o'rtasidagi murakkab o'zaro ta'sirlar. eski, mezbon hujayra va koinfektsion fag lambda (24, 44, 63, 72, 80) va boshqalar. Based on the results of screens of coliforms in Paris and Los Angeles (23, 53) and the Dhillons' extensive work in Hong Kong (40), it became clear that—to the extent that modern notions of segmental evolution (57) allow it— P2 and lambda are representative of two main groups of temperate phages for such bacteria. Within the P2-like group, phage 186 was studied in great genetic and molecular detail by Barry Egan and his students (41, 92) phage 299 was studied to a lesser extent by E. I. Golub (45). More recently, other phages obviously related to P2 have been encountered in several other species besides E. coli and as defective prophages in DNA sequencing studies (34).

The above recollections seem to indicate that at about the middle of the last century, starting before the formulation of the DNA structure and independently of the introduction and diffusion of “molecular” methods of analysis, there was a confluence of the rigorous approach of phage work with more traditional bacteriological perspectives, energized by the new remarkable findings about gene transfer. Studies of lysogeny were rather central in this process of broadening interest with respect to problems and materials. As a result, several new 𠇎xperimental systems” à la Rheinberger were developed in the fifties and sixties, lambda being the most successful. One is tempted to generalize these observations and suggest that it is the way of scientific progress to alternate between periods of broad and somewhat haphazard exploration and periods of highly focused in-depth analysis of particular problems or materials. On the one hand, as Francis Crick once wrote (38), �w molecular biologists would care to be caught studying the colour of butterflies' wings… .” The tendency in molecular biology (as it was, mutatis mutandis, in classical genetics) is for one to analyze the experimental material to the lowest possible structural level and thus invest heavily in one's system. On the other hand, the naturalist looks open mindedly for what may happen to be there and how it might be related to what has already been seen: in a way, he scouts for new experimental systems.

A comment may be made concerning induction: not lambda's, that of philosophers. It is hard to see how our intelligent species would have ever evolved without trusting induction, yet there are everyday examples of the risks of excessive reliance upon inductive predictions. dan keyin B. megaterium phage, a phage in Pseudomonas, and lambda in K-12 were found to be inducible by UV light, it was hard to believe that P2 was not. How do you prove a negative? Similarly, after seeing that both lambda and P2 prophages were present in single copies, physically integrated in the bacterial chromosome at specific sites, it was very surprising when P1 was found to be present as one unattached copy and yet be regularly transmitted to the bacterial progeny without losses (50) or Mu to be capable of inserting itself anywhere in the chromosome (85, 86). Perhaps most surprising, after the early efforts had convinced everyone that lysogens produce phage through the lysis of individual cells, was the discovery that filamentous phages are indeed continuously secreted by growing cells, without lysis (49).

Postscript. My first paper on lysogeny (5), describing the modified single-burst experiment and the isolation of P1, P2, and P3, also contained the formula of the LB medium which I had concocted in order to optimize Shigella growth and plaque formation. Its use has since become very popular. The acronym has been variously interpreted, perhaps flatteringly, but incorrectly, as Luria broth, Lennox broth, or Luria-Bertani medium. For the historical record, the abbreviation LB was intended to stand for “lysogeny broth.”


PDusk and pDawn

The pDusk and pDawn plasmids are engineered DNA systems that allow someone to control gene expression using light. Both pDusk and pDawn make R e d Fluorescent Protein (RFP), wh ich makes the colonies reddish in color. The difference is that pDawn turns on the RFP gene when exposed to light, while pDusk will only make RFP when kept in the dark . The pDawn system allows you to control the expression of RFP in the light. This kit teaches you many basic molecular biology techniques, while also giving you the ability to perform cool experiments using light to control gene expression.

The pDusk and pDawn systems are activated by blue light but since most white light contains blue light any sort of ambient light should work.

  • 1 hour Make plates (set aside more time if it's your first time making plates)
  • streak out bacteria onto an LB Agar plate (takes

Day of experiment overview

    Mix bacteria t ogether with the plasmids, and transformation mix (takes

Incubate and wait for growth

1 hour, but leave more time if it&rsquos your first time)

Step by step walk-through of making plates with photos at: https://goo.gl/7yzpA1

Agar plates provide a solid media nutrient source for bacteri a to grow on. The standard media that is used is LB (Luria Broth, Lysogeny Broth, or Luria Bertani Broth). This contains a carbon source, a nitrogen source, and salt (many strains of bacteria like salt!).

The top of the plate is the larger part.

  1. Find the tube labelled &ldquoLB Agar M edia &rdquo dump its contents into the 250mL glass bottle. (You will need to make plates out of each kind of media . Rinse bottle between media. )
  1. Using the 50mL conical tube labelled &ldquo Tube for Measuring &rdquo, measure and add 150mL of water to the glass bottle.
  1. Making agar is like making jello-- heat the agar to dissolve it, then it will solidify when it cools. Heat the bottle in the microwave for 30 seconds at a time, being careful not to let the bottle boil over. DO NOT SCREW THE LID DOWN TIGHT! (just place it on top and give it a slight turn).
  1. The media is completely dissolved when the liquid looks yel low . This should take about 2 -3 minutes total of microwaving. Take the bottle out , (caution contents hot), and let it cool until you are able to touch it without much discomfort. This will take 20-30 minutes.
  1. While the bottle remains somewhat warm, pour the plates. One at a time, remove the lid of 7 plates and pour just enough of the LB agar from the bottle to cover the bottom half of the plate. Put the lid back on.

Making Competent Bacterial Cells for Transformation

&lsquoCompetent&rsquo means the bacterial cells are able to intake foreign DNA. The cells&rsquo walls normally prevent things from entering , but mixing the bacteria with chemicals and salts that change the cells&rsquo walls, allow the cells take up DNA from the environment. This process is called a &lsquotransformation&rsquo. We put all the components into synthetic DNA to trick the bacteria into thinking that our DNA is its own DNA , so they make the RFP.

The bacterial transformation mix contains:

10% Polyethylene Glycol(PEG) 8000

PEG 8000 is thought to play several different roles in transformation, though nobody really knows for certain. Since both DNA and cell walls are negatively charged, they reject each other. PEG 8000 is thought to function by shielding the charge of the DNA, thereby making it easier to permeate the cell wall. PEG 8000 is also thought to help transport the DNA into the cell, as well as make the cell membrane itself more porous.

5% Dimethyl Sulfoxide (DMSO)

DMSO is sometimes used to treat ailments in humans. In a transformation it is thought to permeabilize the cell wall. Also, sometimes DNA folds into complex structures that make it more difficult to pass through the cell wall. DMSO also might help to break these DNA structures down.

25mM Calcium Chloride(CaCl 2 )

Similarly to PEG 8000, CaCl 2 is thought to shield and neutralize the negative charge of DNA, thereby making it more likely to enter into the cell.

    Take one of the tubes of dried E. coli BL21, add water to the top and shake till it is all dissolved. Next, using your pipette put 100uL of the bacteria solution onto a new LB plate you made and using an inoculation loop gently spread or &ldquostreak&rdquo the bacteria. Let the plate grow overnight

  1. Use a syringe to transfer 0. 1 mL of Transformation mix to a microcentrifuge tube with pDawn . Make sure you mix the transformation mix with the tiny droplet of pDawn DNA in the microcentrifuge tube. Repeat with with pDusk DNA using another syringe. (Syringes can be washed with soap and water for reuse!!)
  1. Using an inoculation loop, gently scrape an area of the size of a pencil eraser of bacteria off of your fresh plate and mix it into the transformation p Dawn mix. Mix until any big clumps have disappeared. Twirling the inoculation loop can work, but avoid making bubbles. Repeat with pDusk using a clean inoculation loop.
  1. Make a warm water bath for the next step. The water should be 42ºC (108ºF) water. You can approximate this temperature by using water that is warm, but comfortable enough such that you can still keep you hand in it.
  1. Add 1.5 mL of room temperature water (or fill to the top) to one of the LB media microcentrifuge tubes and shake to dissolve the LB.
  1. Using a clean syringe , add 0.5 mL of LB media to your competent cell mixture containing your DNA.
  1. Incubate the tube at 37 º C (99 º F) for 2 hour or 4 hours at room temperature. This step allows to bacteria to recover and replicate the DNA and perform the engineering process. Take an LB/ Kan plate out of the fridge and bring them to room temperature
  1. Pour half of the LB/competent cell mix to your LB/Kan plates and gently spread the bacteria around the plate with an inoculation loop. L et dry for 10 minutes before putting the lid back on.
  1. Flip the plate upside down to prevent condensation from forming and dripping onto your bacteria.
  1. Incubate the plate at room temperature for 24-48 hours. Keep pDusk in a dark place or wrap the plate with tin foil.
  1. If your transformation was successful, you should have a plate that looks like the one below. If you kept it in the dark the pDusk plate should look a little red. If you kept it in the light the pDawn plate should look red. If not, give it another shot, Science doesn&rsquot always work on the first try. Also, feel free to contact us at [email protected] and we will help you troubleshoot.

Now that you have colonies growing, you can do a couple of tests to see how pDusk and pDawn behave differently in light and in dark conditions.


Tarkibi

The most common growth media for microorganisms are nutrient broths (liquid nutrient medium) or lysogeny broth medium. Liquid media are often mixed with agar and poured via a sterile media dispenser into Petri dishes to solidify. These agar plates provide a solid medium on which microbes may be cultured. They remain solid, as very few bacteria are able to decompose agar (the exception being some species in the genera: Cytophaga, Flavobacterium, Bacillus, Pseudomonas, va Alcaligenes). Bacteria grown in liquid cultures often form colloidal suspensions. [4] [5]

The difference between growth media used for cell culture and those used for microbiological culture is that cells derived from whole organisms and grown in culture often cannot grow without the addition of, for instance, hormones or growth factors which usually occur in vivo. [6] In the case of animal cells, this difficulty is often addressed by the addition of blood serum or a synthetic serum replacement to the medium. In the case of microorganisms, no such limitations exist, as they are often unicellular organisms. One other major difference is that animal cells in culture are often grown on a flat surface to which they attach, and the medium is provided in a liquid form, which covers the cells. In contrast, bacteria such as Escherichia coli may be grown on solid or in liquid media.

An important distinction between growth media types is that of defined versus undefined media. [1] A defined medium will have known quantities of all ingredients. For microorganisms, they consist of providing trace elements and vitamins required by the microbe and especially defined carbon and nitrogen sources. Glucose or glycerol are often used as carbon sources, and ammonium salts or nitrates as inorganic nitrogen sources. An undefined medium has some complex ingredients, such as yeast extract or casein hydrolysate, which consist of a mixture of many chemical species in unknown proportions. Undefined media are sometimes chosen based on price and sometimes by necessity – some microorganisms have never been cultured on defined media.

A good example of a growth medium is the wort used to make beer. The wort contains all the nutrients required for yeast growth, and under anaerobic conditions, alcohol is produced. When the fermentation process is complete, the combination of medium and dormant microbes, now beer, is ready for consumption. The main types are

  • cultural media
  • minimal media
  • selective media
  • differential media
  • transport media
  • indicator media

Culture media Edit

Culture media contain all the elements that most bacteria need for growth and are not selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections.

An undefined medium (also known as a basal or complex medium) contains:

  • a carbon source such as glucose
  • suv
  • various salts
  • a source of amino acids and nitrogen (e.g. beef, yeast extract)

This is an undefined medium because the amino-acid source contains a variety of compounds the exact composition is unknown.

A defined medium (also known as chemically defined medium or synthetic medium) is a medium in which

Examples of nutrient media:

Minimal media Edit

A defined medium that has just enough ingredients to support growth is called a "minimal medium". The number of ingredients that must be added to a minimal medium varies enormously depending on which microorganism is being grown. [7] Minimal media are those that contain the minimum nutrients possible for colony growth, generally without the presence of amino acids, and are often used by microbiologists and geneticists to grow "wild-type" microorganisms. Minimal media can also be used to select for or against recombinants or exconjugants.

Minimal medium typically contains:

  • a carbon source, which may be a sugar such as glucose, or a less energy-rich source such as succinate
  • various salts, which may vary among bacteria species and growing conditions these generally provide essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the bacteria to synthesize protein and nucleic acids
  • suv

Supplementary minimal media are minimal media that also contains a single selected agent, usually an amino acid or a sugar. This supplementation allows for the culturing of specific lines of auxotrophic recombinants.


Examples of lysogeny broth in the following topics:

Culture Media

  • The most common growth media nutrient broths (liquid nutrient medium) or LB medium (LysogenyBroth) are liquid.

Plasmids and Lysogeny

  • Both plasmids and lysogeny are used by bacteria and viruses to ensure transfer of genes and nucleic acids for viral reproduction.
  • Lysogeny is the process by which a bacteriophageintegrates its nucleic acids into a host bacterium's genome.
  • Lysogeny is utilized by viruses to ensure the maintenance of viral nucleic acids within the genome of the bacterium host.
  • Lysogeny is one of two major methods of viral reproduction utilized by viruses.
  • An example of a virus which can promote the transformation of bacterium from a nontoxic to toxic strain via lysogeny is the CTXφ virus.

Temperate Bacteriophages: Lambda and P1

  • With phage the term virulent is often used as an antonym to temperate, but more strictly a virulent phage is one that has lost its ability to display lysogeny through mutation, rather than a phage lineage with no genetic potential to ever display lysogeny (which more properly would be described as an obligately lytic phage).
  • Yilda lysogeny, P1 can exist within a bacterial cell as a circular DNA, in that it exists by replicating as if it were a plasmid and does not cause cell death.
  • davomida lysogeny, new phage particles are not produced.
  • A unique feature of phage P1 is that during lysogeny its genome is not incorporated into the bacterial chromosome, as is commonly observed during lysogeny of other bacteriophage.
  • This virus is temperate and may reside within the genome of its host through lysogeny.

History of Microbiology: Hooke, van Leeuwenhoek, and Cohn

  • Lazzaro Spallanzani (1729–1799) found that boiling broth would sterilise it and kill any microorganisms in it.
  • He also found that new microorganisms could settle only in a broth agar broth was exposed to the air.
  • By boiling the broth beforehand, Pasteur ensured that no microorganisms survived within the broths at the beginning of his experiment.
  • Nothing grew in the broths in the course of Pasteur's experiment.
  • This meant that the living organisms that grew in such broths came from outside, as spores on dust, rather than spontaneously generated within the broth.

Pasteur and Spontaneous Generation

  • In summary, Pasteur boiled a meat broth in a flask that had a long neck that curved downward, like a goose.
  • The idea was that the bend in the neck prevented falling particles from reaching the broth, while still allowing the free flow of air.
  • When the flask was turned so that particles could fall down the bends, the broth quickly became clouded .
  • In detail, Pasteur exposed boiled broths to air in vessels that contained a filter to prevent all particles from passing through to the growth medium, and even in vessels with no filter at all, with air being admitted via a long tortuous tube that would not allow dust particles to pass.
  • Nothing grew in the broths unless the flasks were broken open, showing that the living organisms that grew in such broths came from outside, as spores on dust, rather than spontaneously generated within the broth.

The Lytic and Lysogenic Cycles of Bacteriophages

  • Those phages able to undergo lysogeny are known as temperate phages.
  • Even though there are similarities between lysogeny and latency, the term lysogenic cycle is usually reserved to describe bacteriophages.

Pure Culture

  • Another method of bacterial culture is liquid culture, in which the desired bacteria are suspended in liquid broth, a nutrient medium.
  • The experimenter would inoculate liquid broth with bacteria and let it grow overnight (they may use a shaker for uniform growth).

Tissue Culture of Animal Viruses

  • Viruses cannot be grown in standard microbiological broths or on agar plates, instead they have be to cultured inside suitable host cells.

Complex and Synthetic Media

  • Luria Broth as shown here is made with yeast extract, as yeast extract is not completely chemically defined Luria Broth is therefore an undefined media.By Lilly_M [GFDL (http://www.gnu.org/copyleft/fdl.html) or CC-BY-SA-3.0-2.5-2.0-1.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons

Minimal Inhibitory Concentration (MIC)

  • MICs can be determined on plates of solid growth medium (called agar, shown in the "Kirby-Bauer Disk Susceptibility Test" atom) or broth dilution methods (in liquid growth media, shown in ) after a pure culture is isolated.
  • For example, to identify the MIC via broth dilution, identical doses of bacteria are cultured in wells of liquid media containing progressively lower concentrations of the drug.
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VWR Life Science Premixed LB Broth

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Is there a difference between Luria Broth and Lysogeny Broth? - Biologiya

Rich media used for routine culture of E. coli and other bacteria at high cell densities.

1L 5L Komponent
10 g 50 g Tryptone
5 g 25 g Yeast Extract
10 g 50 g NaCl

Add dH2O to final volume. Autoclave. If mixing up large batches and aliquoting, be sure to add exact volumes to final media bottles so that they will be ready for addition of antibiotic to known concentrations.

This recipe is from Miller JH. (1992) A Short Course in Bacterial Genetics. CSHL Press. Handbook Unit 25.5.

Note: Be aware that (1) there are several other formulations that may be called LB but vary the amount of NaCl. Using the wrong one can cause large changes in growth. (2) LB was originally supposed to stand for "Lysogeny Broth" and you may also see it called "Luria Broth" (more about this).

Expected results: E. coli strains grow to 5×10 9 cells/ml final density in LB.

Variant: 0.1×LB is made with 1 g Tryptone, 0.5 g Yeast Extract, and 10 g NaCl per liter. (It is 0.1× in the nutrients, but emas the salt!)


Is there a difference between Luria Broth and Lysogeny Broth? - Biologiya

When it comes to growing bacterial colonies, LB-agar steps up to the plate &ndash but first you have to get it into a gel state (a situation to which its friend agarose can surely relate!) &ldquo-ose&rdquo is an ending that&rsquos usually used to indicate something&rsquos a sugar &ndash and agarose hisoblanadi a sugar &ndash but so is agar! So what&rsquos the difference? They&rsquore related, but they differ by more than just a few letters and those differences make them useful for making gel matrices for different tasks &ndash agarose gels for separating DNA fragments by size and agar gel plates for growing bacteria)

A gar is a fish-like thing and AGAR (aka agar-agar (seriously!)) is a aralash of 2 sugars &ndash agaropectin & the one we&rsquore more familiar with, agaroz. So you get agarose by purifying agar. And to understand why you&rsquod go through that trouble sometimes, but not other times, it helps to know a bit more about what these sugars are.

Agarose a polisaxarid (&ldquopoly&rdquo means many & saccharide&rsquos sugar, so a polysaccharide is a long chain of repeating sugar subunits joined together). It&rsquos an example of a polymer. Polymersare long chains of repeating subunits. Different polymers have subunits of different types (e.g. nucleotide subunits link up to give you the polymers we call DNA or RNA amino acids join to form proteins and monosaccharide sugars chain-ify to give make complex carbs (like agarose!))

Sugars have lots of hydroxyl (-OH) groups (which water happily sticks to, helping you form a gel &ndash an &ldquoinfinitely&rdquo interconnected (like 7° of Kevin bacon) polymer mesh containing water. (more to come)). Individual sugar units (monosaccharides) often adopt ring structures (as is the case in agarose) in which the -OH groups stick out like legs. Sugars can have the same &ldquolinkage&rdquo but have the linked groups sticking out in different ways & we use &ldquoL&rdquo and &ldquoD&rdquo to refer to which direction in space the legs point. More on such stereochemistry here: bit.ly/2Q8Dnax

Monosaccharides can use their -OH&rsquos to link together. Linking 2 gives you a disaccharide . Add a few more and you get oligosaccharides (oligo means few). Link lots and you get a polysaccharide (poly meaning many).

And speaking of many, the multiple -OHs mean there are multiple potential linkage sites (which we specify by which number &ldquoaddresses&rdquo on the sugar rings are joined). You can get &ldquobranching,&rdquo but in agarose, you have linear chains (of about 400 subunits). (Although branches can be introduced using &ldquocrosslinkers&rdquo to strengthen agarose so that it can do things like make size exclusion chromatography (&ldquogel filtration&rdquo) resin which can withstand the high pressures generated in FPLC (Fast performance liquid chromatography).

In agarose, the repeating subunit is a sugar duo (disaccharide) of galactose (D-galactose to be precise) linked to a modified galactose monosaccharide, 3,6-anhydro-L-galactose. We call this duo AGAROBIOSE (Linking a galactose to a glucose gives you lactose, a disaccharide you might be more familiar with).

In galactose, the rings have 6 sides with 4 -OH legs, 5 -H legs, & 1 methylhydroxyl (-CH₂OH) leg. We often don&rsquot draw the &ldquo-H&rdquo legs because they hide the more interesting legs that are capable of reacting. In agarose&rsquos modified galactose, 2 of the -OH groups have linked up & kicked out a hydrogen (&ldquoanhydro&rdquo) so that the methyl hydroxyl arm is bridged to an -OH arm forming a &ldquobridge&rdquo over the ring.

About 2/3 of agar is agarose but, agar also contains AGAROPECTIN. It&rsquos really similar (it has alternating D & L galactoses) BUT many of those galactoses have modifications. There are several different modifications including adding sulfate(s), pyruvic acid, or methyl groups, or sneaking in one of those linked-leg versions like&rsquos in agarose.

Unlike the consistent repeating nature of AGAROSE, it&rsquos only the alternating D- & L- galactose &ldquoskeleton&rdquo that&rsquos consistent in agaropectin. Its modifications are scattered throughout the chain (for instance,

every 10th is attached to a sulfate through an -O-sulfate linkage, but that&rsquos just on average, and it&rsquos not likely they&rsquore precisely, evenly, distributed). And, while the chains tend to be shorter than the agarose chains, their length is also variable, so, agaropectin&rsquos really quite a mix & you never know quite what you&rsquore gonna get!

Why use one over the other?

DNA has a lot of negatively-charged phosphate groups (phosphorus surrounded by 4 oxygens). This will serve the basis for them moving through the gel towards the positive end. So we need the gel to be neutral.

Agar has a lot of sulphate groups (sulfur surrounded by oxygens). These are also negatively-charged, so they can interfere with how the DNA moves through the gel. So it would make a bad matrix for electrophoresis.

BUT agarose is neutral, making a good matrix for electrophoresis.

BUT agarose is also more expensive because it has to be purified. So if you don&rsquot need to worry about the physical charge issue, might as well use something that has a lower *monetary* charge! Because agar requires less processing, its cheaper & perfect for use as a matrix to hold bacteria food!.

In agar plates, it&rsquos not the agar itself that&rsquos providing nutrients. That&rsquos one of the great things about agar &ndash *most* bacteria can&rsquot eat it. Instead, the agar just provides &ldquoscaffolding&rdquo to house the nutrients the bacteria need. And often those nutrients are provided through a liquid bacterial food &ldquogrowth media&rdquo called LB, which, no matter what your textbooks might say, (originally at least) stands for Lysogeny Broth. Sometimes initials for Luria, Lennox, or Luria & Bertani get credit for the name, but it really stands for LYSOGENY BROTH and its recipe was first published (by Giuseppe Bertani) in 1951. He was using it when studying lysogeny (a process where a bacteria-infecting virus called a bacteriophage (&ldquophage&rdquo) inserts its own DNA into a bacteria&rsquos DNA & bides its time until conditions are right for entering the lytic phage where it cuts itself out, makes lots of copies and bursts open the cell) http://bit.ly/2HLuB1S

The point of LB is basically to give the bacteria what they need to grow, divide, and do what we want (like make copies of DNA we put in them and/or make copies of proteins using instructions from DNA we put in them). And to do it cheaply.

Note: For the protein-making and large-scale DNA-making, we grow bacteria in straight-up LB liquid &ndash &ldquoin suspension&rdquo with shaking &ndash but when we need to isolate specific groups of bacteria that are all derived from the same &ldquoparent cell&rdquo and thus genetically-identical, we embed that LB into a gel so that different genetically-distinct &ldquocolonies&rdquo don&rsquot intermingle, but instead grow as gloopy dots. If you want to learn more about various media for the suspension stuff check out this post http://bit.ly/bacterialmedia but today I&rsquom going to focus on the gel-trapped form.

We don&rsquot give the bacteria 5-star cuisine. Instead, we want to spend as little money as possible while still giving them the nutrients they need. At a minimum, we need to give them a source of energy, something they can break down (catabolize) to make ATP &ndash such things can be sugars, proteins, fats. In addition to breaking things down, they need to be able to make things like proteins and DNA (do the anabolic part of metabolism). This requires nutrients that provide the elements needed like carbon and nitrogen, which thankfully you can get with a simple recipe that&rsquos sufficient for lots of bacteria.

There are 3 main components (though 2 of those components themselves have a lot of components.

  • TRYPTONE -> this is a mix of peptides formed by the digesting a protein called casein with pancreatic enzyme -> this provides amino acids the bacteria can use to make new proteins
  • YEAST EXTRACT -> this &ldquoautolysate&rdquo of yeast is basically just whatever happened to be in yeast (organic compounds including vitamins, trace elements, etc.) &ndash and if it was good enough for the yeast&hellip
  • SODIUM CHLORIDE (NaCl)(table salt) -> allows for osmotic balance, transport, etc.

A few of the major LB formulations are the &ldquoMiller,&rdquo &ldquoLennox,&rdquo & &ldquoLuria&rdquo versions & they differ in the amount of salt they have. Miller & Bertani drown the bacteria in NaCl (10g/L) whereas Lennox just uses 5g/L and Luria just 0.5g/L -> such low salt recipes are good if you&rsquore using a salt-sensitive antibiotic. in the original paper, Bertani also added glucose, but most later recipes leave it out.

And speaking of leaving things out, we need to make sure we &ldquoleave out&rdquo bacteria we don&rsquot want, which we can do by &ldquoselecting for&rdquo the bacteria we qilmoq want using selection media, which contain things like antibiotics etc. that suppress the growth of things you don&rsquot want to grow. For example, when we put genes into bacteria, we normally do it in the form of circular pieces of DNA called plasmids. We design those plasmids to also have an antibiotic resistance gene, so we can spike the food with that antibiotic and it can still grow, but other stuff can&rsquot http://bit.ly/2tcW4ky

There&rsquos also differential media &ndash this allows for &ldquoscreening&rdquo as opposed to &ldquoselection&rdquo &ndash you don&rsquot keep things from growing, but you change how they appear &ndash for example, we use X-gal for blue-white screening http://bit.ly/2MxNPs2

After I put a plasmid with my gene into bacteria and get colonies, I pick a few of those colonies and put them in liquid LB (with antibiotic) to grow overnight to make lots of copies of the plasmid, then I can purify out those copies and send them for sequencing to check for typos before l enter the &ldquoexpression prep&rdquo part.

Regardless of what media you use, you need it to be sterile. So you autoclave it (stick it in a really hot, high pressure dishwasher) -> make sure the bottles aren&rsquot sealed tight or they&rsquoll explode (thankfully I haven&rsquot made this mistake) &ndash and don&rsquot re-tighten the lids until the bottles have cooled of or the lids will get stuck (I *have* made this one). Another mistake not to make -> don&rsquot add antibiotics before autoclaving, or you&rsquoll inactivate them. We usually don&rsquot add it until right before we&rsquore ready to use it.

In undergrad, I made all my own media, but here, we use so much of it, we have a &ldquomedia-maker&rdquo lab technician who&rsquos amazing and makes & sterilizes our bacterial growth media. You know something&rsquos been through an autoclave if the lines on its autoclave tape are black &ndash the tape is temperature-sensitive so it color-changes when it gets really hot. I really want to design a line of joke and/or trivia messaged autoclave tape &ndash so if the whole teaching thing doesn&rsquot work out, I guess I have a back up!


Videoni tomosha qiling: LB Agar. Luria Bertani medium. Lysogeny broth (Dekabr 2021).