Part:BBa_K1025007
Bitter Defender Part（RBS_B0032）
Bitter Defender Part(RBS_B0032) is a plasmid constructed by 2013 iGEM Tsinghua-E team which is used for providing of selection pressure for the evolution and enrichment of tryptophan overproduction microorganism phenotype. It is almost the same with Bitter Defender Part(RBS_B0030)（BBa_K1025006） except for the second RBS.
Usage and Biology
This selection pressure was based on the tryptophan dependent tetracycline antiporter expression which functioned in tetracycline culture condition. It is achieved by cloning E.coli tetracycline antiporter gene (tetA) downstream of our previously constructed tryptophan biosensor（BBa_K1025005） which is controlled by tac promoter between NcoI and BamHI restriction sites in pTrc99A vector. We constructed three bitter pressure part by utilizing three different RBS upstream of tetA gene. We transformed these vectors into three previously engineered E.coli with different tryptophan productivity (unpublished data).
Functional Parameters
The tryptophan productivity of these nine strains (three RBS and three previous productivities) was further confirmed by HPLC after culture in M9YE medium for nearly 25h after 0.1mM IPTG induction.It was clear that transformation of our engineered bitter defender parts didn’t significantly influence original strains’ tryptophan relative productivity. Hence, we further tested these three parts’ performance by measuring the growth rate of these nine strains in different tetracycline concentration. The final results were shown as growth dynamics below.
As shown here, strains carrying our bitter defender part with RBS B0030 and B0032 showed good tryptophan dependent growth property within the first 15h after culture. Further, as the increase of tetracycline, the selection pressure increased and the growth rate of strains decreased. However, with increased selection pressure, especially for B0032, it could be observed that with time window of about 15h, strains with higher tryptophan overproduction showed much higher growth rate with the growth of tryptophan non producer trp000 nearly inhibited by high concentration of tetracycline. However, it was confused to find that strains trp000 carrying bitter pressure part vector tended to grow beyond this time window. That was hypothesized to be due to the inactivity of tetracycline.Another obvious phenomenon was such tryptophan dependent growth difference was more significant when IPTG induction was conducted.Thus, according to the result of tryptophan dependent growth, we designed the evolution protocol for tryptophan overproduction strains.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 616
Illegal NgoMIV site found at 691
Illegal NgoMIV site found at 721
Illegal NgoMIV site found at 941
Illegal NgoMIV site found at 1059
Illegal NgoMIV site found at 1326
Illegal NgoMIV site found at 1521 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1748
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