1 Registry Star
Part:BBa_F2620:Stability/Mutant
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Description
Device failure was caused by the accumulation of a mutant that had a deletion between two 143bp homologous regions in BBa_T9002. Cells harboring the truncated device grew faster than cells harboring the full device under high input conditions, presumably because the mutant did not produce GFP and hence had a reduced demand. Repeating the stability experiment indicated that device failure occurred after a fixed number of doublings. This was a non-intuitive result as we expected that a mutation that conferred a selective advantage would occur at a random time and hence that the time at which that mutant would take over the population would be random. The consistency in device failure time 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 within individual cells rather than across a population of cells. BBa_T9002 was carried on a multi-copy plasmid, pSB3K3 (~60 per cell), and it is conceivable that each cell in the long-term glycerol stock contains a heterogeneous plasmid population containing both full-length and deleted copies of BBa_T9002.
Analysis
Lane 1: 2-log ladder (NEB)
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
Lanes 11, 19, 21, 24:HindIII (NEB) digest of the PCR product in lanes 10, 18, 20, 22.
Lane 1 (bottom row): 2-log ladder (NEB)
Lanes 2, 4, 6:PCR of purified DNA from Lane 2 of the gel shown in Figure 1
Lanes 3, 5, 7:HindIII digest of the PCR product of purified DNA from Lane 2 of the gel shown in Figure 1
Lanes 8, 10:PCR of purified DNA from Lane 8 of the gel shown in Figure 1
Lanes 9, 11:HindIII digest of the PCR product of purified DNA from Lane 8 of the gel shown in Figure 1
We first analyzed the sequence trace data of purified BBa_T9002 DNA but could not find evidence of the presence of the mutant plasmid. We next tested for the presence of the mutant plasmid in the long-term stock by PCR analysis.
We performed a colony PCR on 4 colonies from a freshly streaked plate of MG1655 harboring BBa_T9002. Colonies were picked and diluted in 30 µL of water. 1µL of that cell suspension was added to 1 µL 10X Thermo Pol Buffer (NEB), 4 pM primer VF2 (https://parts.igem.org), 4 pM primer VR (https://parts.igem.org), 0.5 U Taq Polymerase (NEB), 0.25 µM each dNTP and water to 10 µL. This mixture was thermocycled as follows: 95°C for 6 minutes, 25-35 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 70 s, 72°C for 10 minutes. The PCR product was analyzed via gel electrophoresis (9 V/cm 1% agarose in TAE) and the resulting gel is shown in Figure 1. The strongest band for the four colonies was at 2200bp, 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 gel electrophoresis (Figure 2). On the bottom row, lanes 2, 4, and 6 are the results of PCR reactions using different amounts of template DNA extracted from lane 2 of Figure 1. In each case, 15μl of the 50μl PCR reaction were loaded into the gel. Lanes 8 and 10 are the equivalent PCR products using the DNA extracted from Lane 8 of the gel shown in Figure 1. 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 1300bp band is very faint in these lanes and a band exists just above 1000bp and at 270bp which are the expected length of the fragments of the mutant device PCR product digested with HindIII. The origin of the very bright bands at 600bp and 250bp are not known, although the same bands were evident in the colony PCR products also (Figure 1).
This analysis adds weight to the hyposthesis that the plasmid bearing the mutant device is present in low numbers in the long-term stock of the device.