Part:BBa_K3100140

Acid tolerant working part

Usage and Biology:

We have built our acid tolerant working part which insert different Toehold Switchs before the acid tolerant genes to activate their expression in response to cognate RNAs (Trigger RNAs) with arbitrary sequences (Fig. 1). In our VerProS system, this kind of composite parts named working parts. The acid tolerant working part play an important role in VerProS system which can fine regulation of 4 acid tolerant factors. After screening, we can obtain the strongest acid-tolerant strain.

VerProS pool (Fig. 2) can simultaneously optimize up to four genes (in a vector, pACYC184-working part) 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. At the same time, as the Fusion Sites of the BsaI enzyme were specific designed, if we need to optimize up to other 4 genes, we only need to add the Fusion Sites to both ends of the genes, with the same assembly method can easy construct to a new working part and optimize up to new genes.

Fig, 1: Acid tolerant working part<center>
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Fig.2: VerProS Pool

Characterization:

1. Construct acid tolerant working part:
The recognition and specific recombination sites of the BsaI enzyme were added to both ends of the gene and Toehold Switches. Then the plasmids shown in the following figure were constructed s. Finally, Golden Gate assembly was used to construct the plasmids together with the vector plasmid with ccdB into the final acid tolerant working part.

Fig.3 : construct the 10 plasmids by gene synthesis or Gibson Assembly.

Fig 4: construct acid tolerant working part by Golden Gate Assembly

We completed the construction of pACYC184- acid tolerant working part with the golden gate assembly (fig. 3). And we have successfully integrated it into the chassis system.

Fig.5: Colony PCR of pACYC184- acid tolerant working part

2. 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 (figure4A). 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 (figure 4B).

Figure6. 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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 3804
  Illegal PstI site found at 3256
  Illegal PstI site found at 4176
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3804
  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 4323
  Illegal PstI site found at 3256
  Illegal PstI site found at 4176
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3804
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 3804
  Illegal PstI site found at 3256
  Illegal PstI site found at 4176
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 3804
  Illegal PstI site found at 3256
  Illegal PstI site found at 4176
  Illegal AgeI site found at 2187
  Illegal AgeI site found at 2251
  Illegal AgeI site found at 3516
  
• 1000
  COMPATIBLE WITH RFC[1000]