A student wants to construct a plasmid 'BacLight', that when transformed into a bacterium will produce light IF AND ONLY IF the sugar lactose is present in the medium. For this, she uses the following DNA sequences: promoter of repressor (Pi), promoter of lacZ(Pz), operator (O) and repressor gene lacI from the lactose operon; and a structural gene X, which encodes a protein that produces light. The different DNA elements (II-V) and a vector (I) were digested with restricted enzymes and ligated to produce the plasmid. The DNAs used along with restriction enzyme sites at their ends are given. Slash(/) in the sequence represents the point at which the restriction enzyme cuts the DNA.
I: 5′-(G/GATCC)-Vector-(T/CTAGA)-3′
II: 5′-(T/CTAGA)-Pi-lacI-(G/GATCC)-3′
III: 5′-(T/CTAGA)-Pz-(G/CTAGC)-3′
IV: 5′-(G/CTAGC)-O-(A/CTAGT)-3′
V: 5′-(A/CTAGT)-Gene X-(T/CTAGA)-3′
Choose the correct sequence of DNAs that can possibly be ligated to produce the plasmid 'BacLight'.
A student wants to construct a plasmid 'BacLight', that when transformed into a bacterium will produce light IF AND ONLY IF the sugar lactose is present in the medium. For this, she uses the following DNA sequences: promoter of repressor (Pi), promoter of lacZ(Pz), operator (O) and repressor gene lacI from the lactose operon; and a structural gene X, which encodes a protein that produces light. The different DNA elements (II-V) and a vector (I) were digested with restricted enzymes and ligated to produce the plasmid. The DNAs used along with restriction enzyme sites at their ends are given. Slash(/) in the sequence represents the point at which the restriction enzyme cuts the DNA.
I: 5′-(G/GATCC)-Vector-(T/CTAGA)-3′
II: 5′-(T/CTAGA)-Pi-lacI-(G/GATCC)-3′
III: 5′-(T/CTAGA)-Pz-(G/CTAGC)-3′
IV: 5′-(G/CTAGC)-O-(A/CTAGT)-3′
V: 5′-(A/CTAGT)-Gene X-(T/CTAGA)-3′
Choose the correct sequence of DNAs that can possibly be ligated to produce the plasmid 'BacLight'.
The correct option is A Vector-Pz-O-Gene X-Pi-lacI-Vector
The correct sequence of DNAs that can possibly be ligated to produce the plasmid BacLight are Vector-Pz-O-Gene X-Pi-lacI-Vector. In presence of lactose, it will bind to the repressor molecule and will change its shape. Due to this, the repressor molecule will leave the operator site which otherwise was blocking the transcription of the operon. RNA polymerase will then bind to the promoter of LacZ (Pz) and the transcription will proceed. This will result in the production of structural gene X and light will produce. In the absence of lactose, lactose inhibitor produced by LacI gene will bind to the operator region and transcription will not take place. The genes are arranged to keep in mind the restriction sites. 5' end always ligates with 3' end.Thus, the correct answer is option B.
The correct sequence of DNAs that can possibly be ligated to produce the plasmid BacLight are Vector-Pz-O-Gene X-Pi-lacI-Vector. In presence of lactose, it will bind to the repressor molecule and will change its shape. Due to this, the repressor molecule will leave the operator site which otherwise was blocking the transcription of the operon. RNA polymerase will then bind to the promoter of LacZ (Pz) and the transcription will proceed. This will result in the production of structural gene X and light will produce. In the absence of lactose, lactose inhibitor produced by LacI gene will bind to the operator region and transcription will not take place. The genes are arranged to keep in mind the restriction sites. 5' end always ligates with 3' end.
