Team:Tsinghua-E/Result

From 2013.igem.org

Revision as of 02:36, 28 September 2013 by Piggy (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

Safety

Strains

BL21(DE3)(mut-bitter30(+))/pSC101wt-ori-mutD-sfGFP/pTrc99A-trpss-30-tetA

BL21(DE3)(mut-bitter32(+))/pSC101wt-ori-mutD-sfGFP/pTrc99A-trpss-32-tetA

Negative control：

BL21(DE3) pSC101wt-ori-mutD-sfGFP/pTrc99A-trpss-30-tetA

BL21(DE3) pSC101wt-ori-mutD-sfGFP/pTrc99A-trpss-32-tetA

Each evolution was conducted in three parallel with only one parallel of negative control.

Results

By using the parts constructed by us, following the protocol described, we conducted the continuous evolution derived by mutD based mutation for two E. Coli BL21 (DE3) strains carrying bitter defender part 30 and 32. The results were shown below:

Table1 Titer increase dynamics of different strains after different rounds of evolution (rbs30)

	1st generation	2nd generation	3rd generation
Control (only have mutation part)	No growth	No growth	No growth
Bitter defender part rbs30 (mg/L)	0.791	3.578	3.119
Bitter defender part rbs30/mutation part (mg/L)	0.500 0.741 0.500	0.919 3.748	2.245 3.482 2.245

Table2 Titer increase dynamics of different strains after different rounds of evolution (rbs32)

	1st generation	2nd generation	3rd generation
Control (only have mutation part)	No growth	No growth	No growth
Bitter defender part rbs32 (mg/L)	0.673	1.063	3.433
Bitter defender part rbs32/mutation part (mg/L)	3.142 0.913 0.718	0.766 3.049 4.257	2.540 3.849 3.614

It was obvious that tryptophan productivity evolution indeed occurred with the increase productivity trend along the time course compared with the control group. From the wild type E. Coli BL21 (DE3) with nearly zero productivity, this sensor-based evolution approach successfully increase the productivity to about 3mg/L after only three rounds with totally irrationally knowledge-free fashion. Meanwhile, the control group without bitter defender part showed no growth during the three rounds of evolution, which gave strong proof that it was our constructed system which contributed to the evolution process. However, the strains carrying mutation part showed no difference in final tryptophan titer than that of strains with naturally occurring mutation. According to the report of Leconte A. M.et. al1, in certain conditions, higher mutation rate contributed significantly to the final desired phenotype obtainment along the designed evolution process of biomolecule. However, for our whole-cell level evolution process, considering the inhibition caused by mutation to cell growth, it was possible that in our designed conditions the fitness of higher mutation was not such significant compared with inhibition in fitness. Thus, it was worthy considering resetting our evolution conditions to find the best ones for fast evolution derived by super mutator in the future work. We further plated the evolved strains on agar plate, and picked 24 single colonies to culture in 24-well plate for 48h and detected tryptophon production by HPLC.