Qhov sib txawv tseem ceeb ntawm Golden Gate thiab Gibson Assembly yog tias Golden Gate tso siab rau qhov muaj cov chaw txwv tsis pub dhau ib ntu uas yuav tsum tau cloned, thaum Gibson Assembly tsis cia siab rau qhov muaj cov chaw txwv tsis pub dhau ib ntus rau tau cloned.
Ntau xyoo dhau los, cov kws tshawb fawb molecular tau tsim ntau yam txheej txheem txheej txheem uas tso cai kom yooj yim sib sau ua ke ntawm ntau yam DNA tawg rau hauv ib qho. Yog li ntawd, cloning txoj kev xws li txwv Enzyme Ligation, Gateway Cloning, Gibson Assembly, Golden Gate Assembly, thiab TOPO Cloning yog siv dav los ntawm cov kws tshawb fawb hauv kev sim kev tshawb fawb. Yog li ntawd, Golden Gate thiab Gibson Assembly yog ob txoj hauv kev tsim cov molecular cloning uas tso cai rau kev sib dhos ntawm ntau cov DNA tawg rau hauv ib qho.
Golden Gate Assembly yog dab tsi?
Golden Gate yog ib txoj hauv kev molecular cloning uas yooj yim rau kev sib dhos ntawm ntau DNA tawg rau hauv ib thooj. Txoj kev no tso siab rau qhov muaj cov chaw txwv tsis pub dhau ib ntu uas yuav tsum tau cloned. Nws originated nyob rau hauv 1996. Cov txheej txheem no siv hom IIS txwv enzymes thiab T4 DNA ligase. Cov enzymes no txiav DNA sab nraum qhov chaw lees paub. Lawv tuaj yeem tsim cov tsis muaj palindromic overhangs. Yog li ntawd, ntau qhov tawg ntawm DNA tuaj yeem sib sau ua ke siv kev sib txuas ntawm kev sib txuas ua ke.
Daim duab 01: Golden Gate
Golden Gate Assembly Procedure
Golden Gate txheej txheem muaj peb kauj ruam tseem ceeb hauv nws cov txheej txheem:
- creation ntawm overhangs nyob rau hauv lub cloning vector,
- sib dhos ntawm ntau qhov DNA ntxig los ntawm kev siv cov ntu ntu tshwj xeeb hauv overhangs, thiab
- ligation.
Golden Gate sib dhos siv cov voj voog cloning vectors (destination vector). Tsis tas li ntawd, nyob rau hauv txoj kev no, qhov chaw txwv yog tshem tawm ntawm cov khoom ligated thiaj li ua tiav kev zom zaub mov thiab ligation ib txhij. Cov txheej txheem thermal voj voog ib txwm rau Golden Gate oscillates ntawm 37 ° C thiab 16 ° C vim tias 37 ° C yog qhov zoo rau kev txwv enzymes thiab 16 ° C yog qhov zoo rau ligases.
Gibson Assembly yog dab tsi?
Gibson Assembly yog ib txoj hauv kev molecular cloning uas tso cai rau kev sib dhos ntawm ntau yam DNA tawg rau hauv ib qho. Tsis zoo li txoj kev Golden Gate, txoj kev no tsis tso siab rau qhov muaj qhov txwv tsis pub dhau ib ntu uas yuav tsum tau cloned. Nws tau pom los ntawm Daniel G. Gibson ntawm J. Craig Venter Institute. Cov txheej txheem no yog ib hom kab ke thiab ligase ywj siab cloning (SLIC) txoj kev.
Daim duab 02: Gibson Assembly
Gibson Cov txheej txheem sib dhos
Gibson Assembly xav tau cov khoom DNA uas muaj 20-40 lub hauv paus khub sib tshooj nrog cov DNA uas nyob ib sab. Overlaps yog ntxiv los ntawm PCR. Tom qab ntawd cov DNA tawg tau ntxiv rau Gibson maser sib tov uas muaj peb enzymes. Cov txheej txheem no yog ua nyob rau hauv isothermal tej yam kev mob (50 ° C rau 1 teev) siv peb yam enzymes: exonuclease, DNA polymerase thiab DNA ligase. Lub exonuclease zom rov qab DNA los ntawm 5' kawg. Yog li, nws tsis inhibit polymerase kev ua haujlwm thiab tso cai rau cov tshuaj tiv thaiv mus rau hauv ib qho txheej txheem. Qhov tshwm sim ib leeg-stranded cheeb tsam ntawm cov DNA uas nyob ib sab tuaj yeem raug muab tshem tawm vim kev sib tshooj sib tshooj. DNA polymerase sau cov qhov khoob los ntawm kev ntxiv nucleotides. Thaum kawg, DNA ligase covalently koom nrog DNA ntawm ntu uas nyob ib sab. Feem ntau, Gibson Assembly siv linearized lo lus uas peb vectors. Ntxiv mus, txoj kev no tuaj yeem sib xyaw ua ke mus txog 15 DNA tawg raws li qhov sib xws.
Zoo sib xws ntawm Golden Gate thiab Gibson Assembly
- Golden Gate thiab Gibson Assembly yog ob txoj hauv kev tsim cov molecular.
- Ob txoj hauv kev tuaj yeem sib sau ntau yam DNA tawg rau hauv ib qho.
- Cov txheej txheem no siv cov vectors.
- Ob txoj hauv kev siv cov tshuab hluav taws xob thermal rau kev sib dhos ntawm ntau yam DNA tawg.
- Lawv siv rau hauv qhov chaw qhia mutagenesis.
Qhov txawv ntawm Golden Gate thiab Gibson Assembly
Golden Gate yog ib txoj hauv kev siv molecular cloning siv nyob rau hauv kev sib dhos ntawm ntau yam DNA tawg mus rau hauv ib qho kev vam khom nyob rau hauv lub xub ntiag ntawm kev txwv qhov chaw nyob rau hauv ib tug tej yam uas yuav tsum tau cloned, thaum Gibson Assembly yog ib tug molecular cloning txoj kev siv nyob rau hauv lub Kev sib sau ua ke ntawm ntau yam DNA tawg rau hauv ib thooj yam tsis muaj kev vam khom ntawm qhov chaw txwv tsis pub dhau ib ntu uas yuav tsum tau cloned. Qhov no yog qhov sib txawv tseem ceeb ntawm Golden Gate thiab Gibson Assembly. Tsis tas li ntawd, Golden Gate txoj kev siv lub voj voog ncig lub voj voog, thaum Gibson Assembly txoj kev siv lub hom phiaj linearized vector.
Cov lus qhia hauv qab no qhia qhov txawv ntawm Golden Gate thiab Gibson Assembly.
Summary – Golden Gate vs Gibson Assembly
Molecular cloning yog ib txoj hauv kev siv los sib sau ua ke DNA molecules recombinant thiab coj lawv rov ua dua nyob rau hauv cov kab mob. Golden Gate thiab Gibson Assembly yog ob txoj hauv kev sib koom ua ke molecular uas tso cai rau kev sib dhos ntawm ntau yam DNA tawg rau hauv ib qho. Txoj kev Golden Gate cia siab rau qhov muaj cov chaw txwv tsis pub dhau ib ntu uas yuav tsum tau cloned, thaum Gibson Assembly tsis cia siab rau qhov muaj cov chaw txwv tsis pub dhau ib ntu uas yuav tsum tau cloned. Yog li, qhov no yog qhov sib txawv tseem ceeb ntawm Golden Gate thiab Gibson Assembly.
