Part:BBa_K4662056

sgRNA to block formate acetyltransferase 1

The sgRNA construtct can be used to block the formate acetyltransferase 1 enzyme when dCas9 is expressed.

Formate acetyltransferase 1 enzyme uses pyruvate as an substrate and plays an important role in central metabolism

This composite parts contains an constitutive anderson promoter BBa_J23100. The crRNA was designed and its efficiency was predicted (65%) with the webpage chopchop and contains the PAM sequence in the beginning of the gene region of the Pyruvate formate-lyase 1-activating. The tracrRNA was implemented from S. pyogenes BBa_K4662031

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Efficiency of synthetic sgRNA and dCAS9

Figure 1: The figure shows the corresponding lactate concentration at each timestep, in both conditions (uninduced and induced with 5ug/ml tetracycline).

The sgRNA construct was assembled with the use of golden gate cloning. The plasmid was then transformed into compentent cells containing the dCas9, with an tetracycline inducible promoter.

The lactate production of each transformed bacterium was measured in two conditions. “uninduced” as a baseline how much lactate is produced normally and “induced” by 5 ug/ml tetracycline. Figure 1 shows nicely that the guide RNA leads to an increase in lactate production. It is evident that the lactate concentration drops in the 19.5h timestep. We hypothesise that upon starvation of the bacteria, the nutrients are not enough to support growth anymore, so the bacteria start to catabolize the lactate within the medium.

With our results we are not able to support or deny the efficiency.

Conclusion

We conclude that the sgRNA design was extremely successful and can be used as a reliable tool to manipulate the central metabolism in the future. The usage of the guide RNAs leads to more available pyruvate in the cell. This allows future users to redirect the flux of e.g.: glycolysis into a desired direction.

Usage and Biology

Part can be used to manipulate central metabolism leading to more available pyruvate in the cell.

Remark

Specific protocols for assembly and testing can be found on our wiki https://2023.igem.wiki/uzurich/experiments

If any questions arise or additional information is needed, we can provide a more extensive list of experiments that we can share if required.