1 Registry Star
Part:BBa_F2620:Stability/Mutant
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Description
Repeating the stability experiment indicated that device failure occurred after a fixed number of doublings. This non-intuitive result could be explained if there was pre-existing genetic variation in the long term stock of the device. Since the experiment was started each time from a single colony, that pre-existing genetic variation must be intra-cellular rather than extracellular. T9002 was carried on a multi-copy plasmid (pSB3K3) and it is conceivable that a small fraction of those were mutants in each cell.
Analysis
Lanes 2, 4, 6, 8:Colony PCR of 4 colonies from a freshly streaked plate of T9002 on pSB3k3 in MG1655
Lanes 3, 5, 7, 9:HindIII (NEB) digest of the PCR product in lanes 2, 4, 6, 8.
Lanes 10, 18, 20, 22:Colony PCR of 4 colonies from a freshly streaked plate of MG1655 containing the mutant T9002 on pSB3k3
Analysis of sequence trace data using purified T9002 DNA did not indicate the presence of the mutant plasmid. We chose to test for the presence of the mutant plasmid in the long term stock by pcr analysis.
In an initial step, we performed a colony PCR on 4 colonies from a freshly streaked plate of T9002. The thermocycling conditions used were - 95C for 1 min, 55C for 30 s, 72C for 70 s repeated 34 times. The PCR product was analyzed via electrophoresis (8 V/cm 1% agarose in TAE) and the resulting gel is shown in Figure 1. The strongest band for the four colonies was at the expected length for T9002 (lanes 2, 4, 6, 8). Multiple shorter bands can also be seen in those lanes, including one band that is the expected length of the T9002 mutant (1300bp). Both BBa_T9002 and the mutant can be digested with HindIII ([http://neb.com NEB]) however digestion of the PCR product with HindIII (3 hrs, 37C) did not conclusively show a shift in the band that might correspond to the mutant device (lanes 3, 5, 7 9).
We purified the DNA from the potential mutant bands in lanes 2 and 8 using a Qiagen gel extraction kit (Qiagen). We performed PCR on the purified DNA using the same thermocycling conditions described above and analyzed the PCR products by electrophoresis (Figure 2). On the bottom row, lanes 2, 4, and 6 are the results of PCR reactions using different amounts of template DNA, 15μl of the 50μl PCR reaction were loaded into the gel. A faint band can be seen at the expected length for the mutant device at 1300bp along with two brighter bands at 600bp and 300bp. 30μl of the PCR products were digested with HindIII and loaded into the gel (lanes 3, 5, and 7). The 1200bp band is very faint in these lanes and a band exists just above 1000bp at at 27bp which are the expected length of the fragments of the mutant device PCR product digested with HindIII.
This analysis adds weight to the hyposthesis that the plamid bearing the mutant device is present, in low numbers in the long-term stock of the device.