Part:BBa_K3100100
T7 Promoter T7-1_Trigger DNA A_T7 terminater
Usage and Biology
BBa_K3100100-BBa_K3100139 are composite parts which are the combination of 4 different Triggers and 10 unique T7 promoters. Through specific design of Fusion Sites, these 40 parts will be randomly combined by Golden Gate assembly. Up to 4 different Triggers driven by 10 unique promoters are expressed from one plasmid and finally a pool called VerProS pool with a storage capacity of 10000 is obtained (Fig. 1). VerProS pool can simultaneously optimize up to four genes in a system. Particularly, this versatile library can be applied to fast optimization in different systems without having to build ad hoc libraries, which can greatly reduce manpower and costs.
Fig. 1 Golden Gate Assembly of VerProS Pool
Characterization
1.construction
Construct the 40 composite part with the puc19 vector by annealing splicing(Fig.2)
Fig. 2: T7 promoter & Trigger DNA composite parts assembly method
pET30a(+) is selected as the plasmid skeleton of the transcription vector, and the lethal gene ccdB is used to select the right splicing transcription plasmid. 40 plasmids containing one promoter and one Trigger each are constructed by free combination of 10 various promoters with 4 different triggers. Finally, these 40 plasmids are randomly combined with pET30a(+)-ccdB plasmids vector through Golden Gate assembly.(Fig.1)These eventually constitute the VerProS pool with a capacity of 10000. And because of standardized recombination sites design, we can easily increase the dynamic range of control and expand the storage capacity of the pool in the future.
2. result
We have completed the construction of 40 plasmids of T7 promoter and trigger combinations. And in order to improve the splicing efficiency of the golden gate assembly, we constructed a final vector of pET30a(+)-ccdB with a 98.9±0.2% mortality rate (Fig.3A). The positive rate of the transformed strain of the golden gate assembly reaction product was 95.2% (Fig. 3B). These are the plates of the product transformed strain (Fig.3C).
Figure3. Construction of VerProS pool (A) These are three plates, pET30 (DH5α), pET30-ccdB (DH5α), pET30-ccdB (transDB3.1). The lethality can be calculated from these plates. (B) Colony PCR gel electrophoresis image. Bands of correct length are seen meaning the construct was successful. (C) The picture of the plates of the product transformed strain.
3. Test of VerProS system
We transformed the VerProS pool to engineering bacteria which have the working part. By enrichment, we obtained the acid tolerant bacteria of the optimal promoter arrangement (fig.4 A). By measuring the growth curve, the bacteria increased by 223.3% compared to the chassis organism.
At the same time, we inoculated the bacteria with different survival conditions in the medium under the pH=4.5 plate. We then measured their de growth curves. And the sequencing of the promoter was obtained by sequencing for modeling (fig.4 B).
Fig.4 : Test of VerProS system (A) The graph of growth curve of VerProS system. The y-axis is OD600. The x-axis is time, unit hour. Two hours after the culture of the bacteria, 1.2 μl of 0.05 M IPTG was added to the medium to induce expression. Error bars indicate the standard error of at least three biological replicates. P<0.05. (B) The graph of growth curve of VerProS system. The y-axis is OD600. The x-axis is time, unit hour. Two hours after the culture of the bacteria, 1.2 μl of 0.05 M IPTG was added to the medium to induce expression.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 54
Illegal EcoRI site found at 70 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 54
Illegal EcoRI site found at 70
Illegal NheI site found at 168 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 54
Illegal EcoRI site found at 70 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 54
Illegal EcoRI site found at 70 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 54
Illegal EcoRI site found at 70 - 1000COMPATIBLE WITH RFC[1000]
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