My First Cloning
Its story of an Indian girl who desired to be a scientist. Those were days of 1970s when we had few science subjects for masters, Botany, Zoology, Chemistry, Physics, M. Sc in Biochemistry had started only in few universities. Belonging to UP, my father was not ready to send me to other state. It was a choice between Allahabad or Kanpur University. Since we lived in Kanpur, choice fell on second option. I joined M. Sc in Botany in Christ Church College, Kanpur University. I finished my M. Sc in 1971 and joined PhD in the same department under Dr A.B. Gupta, an algologist and the then Head of the department. Little I knew what was store for me. In 1972, I got engaged to Brahm who was visiting India from Belgium. He was working in University of Brussels and was on a family visit. Single eligible foreign return bachelors were great attraction those days. As luck would have it, we got engaged and married on 6 February, 1972 and left for Brussels on 16th February. It seemed like heaven. Brahm worked in Radiology Department in University of Brussels. Initially I joined French classes as French was the official language of Belgium. After three months, I joined Laboratoire de Genetique under Professor Rene Thomas. Rene worked on genetics of phage lambda. The lab introduced me to Escherichia coli and phage lambda, the lytic and lysogeny cycle, temperature sensitive mutants of cI repressor protein, assembly of phage DNA into viable phage particle. We returned to India in 1974. Brahm was offered Scientist C position in CSIR-Central Drug Research Institute at Lucknow and I continued my research on DNA dependent RNA polymerase of Escherichia coli and its interaction with phage lambda that I was pursuing in Brussels. In 1978, I was awarded PhD and I decided to go for postdoctoral research in USA along with my 3 year old daughter. I got an offer from Wisconsin University in Prof WaclawSzybalsky lab but had to decline as he was apprehensive about bringing my daughter along. They as working with Epstein virusand he felt my child could be exposed . He recommended me to Prof Susan Gerbi. Susan was working on chromosome organization in fungus fly Sciara coprophila and was looking for someone with experience of handling phage lambda. She intended to make genomic library of Sciara, clone the fragments, sequence them. Cloning was still in initial stage even in USA.
It was a totally different area that I was going to work on. Brahm had some clinical trials going on so we decided that I go ahead with my daughter. I reached Providence, my Professor had come to pick me up at Boston. It was time to start. I was good at growing lambda, that pleased my boss Prof SusanGerbi. Now was the time for isolating DNA. It was Charon 4 phage, I got 4 microgram yield from a litre culture. I was shocked, I repeated, it was worse. Had I forgotten to extract DNA, I took wild type lambda and isolated DNA, 1 mg from a litre. What a relief , so problem was with Charon 4 phage. We decided to drive to Boston to discuss with some friends in Harvard. He said, it’s a sick phage, you have to work out conditions. After several hit and trial, I could get 400 microgram from a litre which was considered pretty good. First landmark.
Now second landmark was development of in vitro packaging system for lambda which consisted of Freeze Thaw lysate, sonicated lysate and Protein A from lambda which cuts at cos site, generating single copy of lambda. I still remember, I started with isolation of Protein A at 8 AM in cold room at 40C Whole process had to be done in cold room. At 5.30 pm, I rushed to day care centre to pick up my daughter, we had quick dinner at McDonald and rushed back to lab. The work had to continue overnight. Radha slept on Coffee table. I finished at 5 AM next morning. I was always a late riser, this experiment gave an opportunity to witness sunrise. I woke up Radha and we went back home.
The experiment was performed. No success. Again we rushed to Boston. For sonicated extract, we had to take lambda lysate, sonicate and do ultracentrifugation at high speed. Since it was a low volume and we were using polyallomer tubes, we will make up the volume with mineral oil. Our friend at Harvard listened to our protocol and said, looks alright except that I hate mineral oil, why don’t you use polycarbonate tube. Back we came, ordered polycarbonate tube. Tubes were on our table next day. Now was time for celebration as experiment was successful.
We could successfully generate genomic library of fungus fly in phage lambda, isolate clones and study band-interband pattern in polytene chromosome. The work was published in JMB. It was a long journey, full of failures, success, trouble shooting but gave me perfect learning. I learnt about restriction enzymes, DNA ligases, bacterial alkaline phosphatase, replacing it with calf alkaline phosphatase with solid practical reasons, generating genomic libraries, DNA sequencing, sequence analysis that too manually. Yes I could not generate more publications but it gave me a solid foundation in recombinant DNA technology which was going to be a game changer in my professional life.