You are a new PhD student in a molecular biology lab at the newly established Xa
ID: 150388 • Letter: Y
Question
You are a new PhD student in a molecular biology lab at the newly established Xanadu Research Institute.The Head of Research at the Institute (Tony George) has given you an initial cloning exercise to gauge your aptitude for research.
Your assignment is to carry out a subcloing scheme, which is described in relation to the diagram below, which depicts the LmrA gene of interest (pink ORF on the right) in a shuttle vector and various restriction sites shown at either end of the LmrA insert.
The central idea of this scheme is simply to remove the LmrA gene from the shuttle vector and to ligate and express the LmrA gene in a suitable expression vector. Xanadu is struggling financially and you can’t just order in any expression vector. You will have to make do with an in-house vector for this task. There are three available vectors, which are: pUC18, pUC19, and pBR322.
Narl 237 Sphl 410 Pstl 416 Accl 419 Xbal 424 amHI 430 6xHis tag LmrA start codon 200bp I 400bp 4200obp amp prom 00bp lacz.a reporter 3800bp amp marker ORF 2 rf(6) 3600bp 1000bp 340obp 200bp_ -3200bp 1400bp .. BR322 origin 3000bp ORF.1 rf(2) 1600bp 2800bp lac prom 1800bp 2600bp 2400bp 2200bp EcoRV 1556 sTo LmrA stop codon EcoRI 2273 Sacl 2271 Kpnl 2265Explanation / Answer
pUC19 is suitable for this subcloning. pUC19 has multiple cloning sites in lacZ alpha fragment. Cloning insert into this site will disrupt the LacZ reading frame and hence the b-galactosidase activity which gives rise to white colonies on X-gal/IPTG plates. The plasmid also shows ampicillin resistance. A gene cloned in frame with the lacZ gene will be expressed as a fusion protein under the lacZ promoter. The Only difference between pUC18 and pUC19 is the orientation of multiple cloning sites. But in this case among these two pUC19 is suitable because the orientation of MCS is suitable for cloning LmrA gene while in pUC18 the orientation of LmrA gene will be reversed.
pBR322 the restriction sites available for cloning are less and most of them are present in the antibiotic resistance gene, cloning in these sites can disrupt the reading frame antibiotic resistance gene and can interfere with the screening process.
To clone the LmrA gene you can digest the gene and vector with any enzymes like SacI, EcoRI, KpnI, PstI, SphI, and BamHI. Make sure that the two enzymes you are using are sufficiently apart from each other. Because enzymes need some space to sit and cut the sequence. If they are too close the cutting will not be efficient.
Run the sample on the gel and purify the fragment of your interest.
Set up the ligation reaction using vector and gene of interest and finally transform the ligation mix.
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