Questions: Biotechnology: Principles and Processes

Q1. Why does genetic engineering avoid the problem that traditional hybridisation faces?

Answer (English): Hybridisation combines the genomes of two parents, so the offspring receive useful genes together with many undesirable ones from both sides. Genetic engineering builds a recombinant DNA molecule instead, using restriction enzymes and a vector, and moves only the chosen gene or a small set of genes into the target organism. The recipient therefore gains the wanted trait without carrying the rest of the donor's unwanted genes.

Hinglish: Hybridisation mein do parents ke saare genes mix ho jaate hain, isliye achhe genes ke saath unwanted genes bhi chale aate hain. Genetic engineering mein recombinant DNA banakar sirf chuna hua gene ya uske chhote set ko target organism mein daala jaata hai, isliye donor ke baaki genes nahi aate.

Q2. State the three basic steps involved in genetically modifying an organism.

Answer (English): The first step is to identify the DNA that carries the desirable gene. The second is to introduce that identified DNA into the host. The third is to maintain the introduced DNA inside the host and transfer it to the host's progeny.

Hinglish: Pehla step: kis DNA mein desired gene hai, use pehchano. Doosra: us DNA ko host mein daalo. Teesra: introduced DNA ko host mein bana raho aur uski progeny tak pahunchao.

Q3. What are sticky ends and why are they important in recombinant DNA technology?

Answer (English): The enzyme cuts slightly off the centre of the palindrome, so short single stranded overhangs are left at the two ends. These overhangs are the sticky ends. They form hydrogen bonds with a complementary cut end, which brings the two DNA pieces together and lets DNA ligase seal the sugar-phosphate backbones into one recombinant molecule. Fragments cut with the same enzyme have matching sticky ends, so they can join end to end.

Hinglish: Restriction enzyme palindrome ke centre se thoda hatkar cut karta hai, jisse ends par chhote single stranded hisse reh jaate hain, inhe sticky ends kehte hain. Ye complementary cut end se hydrogen bonds banaate hain, jisse do DNA pieces paas aate hain aur DNA ligase unhe jod deta hai. Same enzyme se kate fragments ke sticky ends match karte hain, isliye end to end join ho jaate hain.

Q4. Explain the convention used for naming the restriction enzyme EcoRI.

Answer (English): The first letter comes from the genus and the next two letters from the species of the bacterium that supplies the enzyme. The letter before the Roman numeral marks the strain, and the Roman numeral shows the order of isolation from that strain. EcoRI therefore comes from Escherichia genus, coli species and strain RY 13, and it is the first enzyme isolated from that strain.

Hinglish: Naam ka pehla letter genus se aata hai, agle do letters species se, aur Roman numeral se pehle wala letter strain batata hai. Roman numeral yeh batata hai ki us strain se enzyme kis number par alag kiya gaya. EcoRI Escherichia genus, coli species aur strain RY 13 se hai, aur us strain ka pehla enzyme hai.

Q5. What features must a cloning vector have?

Answer (English): A vector needs an origin of replication, so that any DNA linked to it can replicate in the host, and the ori also decides the copy number. It needs a selectable marker, usually an antibiotic resistance gene, to identify and eliminate cells that did not take up the vector. It needs only a few cloning sites, ideally one recognition site for each enzyme in common use, so the insert can be placed without breaking the vector into many pieces.

Hinglish: Vector mein ori hona chahiye taki linked DNA host mein replicate kare, aur ori se copy number bhi decide hota hai. Ek selectable marker chahiye, aksar antibiotic resistance gene, jo transformants ko baaki cells se alag kare. Aur cloning sites kam hone chahiye, behtar hai ek hi site ho, taki foreign DNA daalne par vector ke tukde na banein.

Q6. How are recombinants selected when a cloning vector carries two antibiotic resistance genes?

Answer (English): In pBR322 the foreign DNA goes in at the BamH I site, which sits inside the tetracycline resistance gene, so the insert switches that gene off. Cells that took up a plasmid are first selected on ampicillin, and the transformants are then tested on tetracycline. Recombinants grow on ampicillin but not on tetracycline, because their tetracycline gene is inactive. Non-recombinants grow on both antibiotics.

Hinglish: pBR322 mein foreign DNA BamH I site par tetracycline resistance gene ke andar daalna padta hai, jisse woh gene inactivate ho jaata hai. Pehle ampicillin plate par transformants chunte hain, phir tetracycline plate par test karte hain. Recombinant ampicillin par ugta hai lekin tetracycline par nahi; non-recombinant dono par ugta hai.

Q7. Why must a host cell be made competent, and how is a bacterium made competent?

Answer (English): DNA is hydrophilic and cannot cross the cell membrane by itself, so a bacterium needs help before it can take up a plasmid. A measured dose of a divalent cation such as calcium improves the chance that DNA will cross the cell wall and enter the cell. The cells are then incubated with the recombinant DNA on ice, given a brief heat shock at 42 degrees Celsius, and returned to ice so that the DNA enters the host.

Hinglish: DNA hydrophilic hai, isliye apne aap cell membrane cross nahi kar sakta; bacteria ko pehle taiyar karna padta hai. Calcium jaise divalent cation se cells treat karte hain, jisse DNA cell wall ke pores se andar aane ki efficiency badh jaati hai. Phir recombinant DNA ke saath ice par incubation, 42 degree ka heat shock aur wapas ice par rakhne se DNA host mein chala jaata hai.

Q8. Explain the polymerase chain reaction and the role of the thermostable DNA polymerase in it.

Answer (English): PCR makes many copies of a chosen DNA segment outside a cell. Two sets of primers flank the target and anneal to the two strands. DNA polymerase extends each primer using the nucleotides in the reaction mixture and the template DNA. Each cycle has denaturation of the double stranded DNA, primer annealing and extension of the primers. A thermostable DNA polymerase from Thermus aquaticus is used because it stays active at the high temperature needed for denaturation, and repeated cycles can amplify the segment about a billion times.

Hinglish: PCR cell ke bahar chune hue DNA segment ki bahut copies banata hai. Do sets ke primers target ke flank par anneal hote hain, aur DNA polymerase reaction mixture ke nucleotides se unhe extend karta hai. Har cycle mein denaturation, primer annealing aur extension hoti hai. Thermus aquaticus se mila thermostable polymerase high temperature par bhi active rehta hai, isliye baar baar cycle chala kar arabon copies ban jaati hain.

Q9. Why is large scale production needed, and what does a stirred-tank bioreactor provide?

Answer (English): Small cultures cannot give appreciable amounts of a product, so cells are grown in bioreactors that handle 100 to 1000 litres of culture. A bioreactor converts raw materials biologically into specific products by providing optimum conditions of temperature, pH, substrate, salts, vitamins and oxygen. A stirred-tank bioreactor is cylindrical or has a curved base for good mixing, and its stirrer keeps the contents even and helps oxygen availability. It also has an agitator system, oxygen delivery, foam control, temperature control, pH control and sampling ports.

Hinglish: Chhoti cultures se itna product nahi milta, isliye bioreactor mein 100 se 1000 litre culture ugaya jaata hai. Bioreactor raw material ko biological tarike se product banata hai aur temperature, pH, substrate, salts, vitamins aur oxygen ki optimum conditions deta hai. Stirred-tank bioreactor cylindrical ya curved base wala hota hai, jahan stirrer se mixing aur oxygen availability theek rehti hai; saath mein agitator, oxygen delivery, foam control, temperature aur pH control aur sampling ports hote hain.

Q10. What is downstream processing?

Answer (English): After the biosynthetic stage in a bioreactor, the product has to go through a series of steps before it can be sold as a finished product. Separation and purification of the product are together called downstream processing. The product is then formulated with suitable preservatives, must pass clinical trials when it is a drug, and goes through strict quality control testing. These steps differ from product to product.

Hinglish: Bioreactor ke biosynthetic stage ke baad product ko market ke liye taiyar karne ke liye kai steps se guzarna padta hai. Product ke separation aur purification ko milkar downstream processing kehte hain. Uske baad product ko suitable preservatives ke saath formulate karte hain, drug hone par clinical trials hote hain, aur har product ka strict quality control testing hota hai. Ye steps har product ke liye alag hote hain.

To check how much of this stuck, take the five-question quiz below. Each answer explains the reasoning, so read the explanation even when you get it right.

Quiz: the five question check for this chapter is set from the same topics. Attempt it after the questions above, then revisit any topic page where you lost a mark.