# Recollections of a [PERSON] Disciple
## 2 Making _E. coli_ Minichromosomes and Cell Cycle Analysis  
It turned out that cloning _oriC_ was not as simple a task as I had hoped. A big stumbling block was my attempt to purify the non-replicating drug resistant fragment away from the plasmid's own replication origin. This was achieved using gel electrophoresis to separate the two fragments and then purifying out the desired one. It was inefficient and subject to cross contamination. There were no kits available to speed the process, so I failed many times at this step and at the final step of cell transformation with the ligated chromosomal fragments. Unhappily, my repeated failures were noticed by one of [PERSON] post-docs who, in an effort to be amusing, added a \"cloning report\" to his weekly tongue-in-cheek newsletter (The Flash) on lab group happenings. While I looked forward to his funny and often clever take on the events of the week, the weekly cloning report of \"no progress\" was not particularly uplifting (Figure 2). When several interesting colonies finally appeared on a transformation plate and were shown to carry a new plasmid of the correct size expected for a minichromosome, I was horrified to observe that these plasmids were highly unstable in my _E. coli_ host. This turned out to be due to the carry-over of some genes from the ATP synthetase operon adjacent to _oriC_ on the chromosome, but until I was able to construct deletion derivatives, I grew cultures containing my first minichromosome (pAL1) at very high concentrations of antibiotic to kill off the plasmid-less segregants. Using 0.5 mg/mL kanamycin in the media  
Figure 2: _OriC_ cloning reports from the weekly lab newsletter “The Flash”. See text. seemed ridiculous at the time, but it allowed me to isolate enough minichromosome DNA for future experiments. It is important to note that I was not the first to make an _E. coli_ minichromosome. That honor went to others in the labs of [PERSON] and [PERSON]/[PERSON], whose ground-breaking work provided the nucleotide sequence of _oriC_ and began the difficult process of identifying the protein recognition motifs [7; 8]. The enormous efforts of [PERSON] and [PERSON] in [PERSON]'s lab then produced an in vitro replication system that was pivotal to dissecting the chromosomal replication machinery [9]. Since our lab was focused on the regulation of DNA replication in living cells, our intent was to measure the replication of minichromosomes during the cell cycle. The obvious question was whether the minimal _oriC_ region was sufficient for proper initiation timing during the cell cycle. The answer would not only reveal important features of the regulatory mechanism, but validate the minichromosome model for future studies, particularly those performed using in vitro systems. While we hoped that periodic minichromosome replication was retained, their moderately high copy number (10-20 copies per cell), despite their instability) did not seem compatible with the properly timed, once-per-cycle regulation of chromosomal _oriC_. We anticipated random replication but did not discount the possibility of periodic replication with initiation timed differently than the chromosome. Rather than working with synchronously growing cells, we based our experimental design on the \"backwards\" baby machine approach [PERSON] and [PERSON] had used previously to study chromosome replication [5], since this would minimize artifacts caused by manipulating the cells. Since minichromosomes replicate in a matter of seconds, we believed that a minimally manipulated system would be important in distinguishing cell cycle-specific replication from random replication throughout the cell cycle. In this \"backwards\" procedure, exponentially growing cultures were pulse-radiolabeled with tritiated thymidine to label any replicating DNA. Then, the entire culture was transferred to a nitrocellulose membrane filter, the unincorporated label was washed out, and baby cells were eluted and collected at 1/10 th generation intervals from the dividing cell population on the surface.