Part:BBa_K4662047

RhlR and LDH level 2

Level 2 plasmid containing constitutive expressed RhlR level 1 plasmid and inducible LasB promoter with the . Strepptococcus Bovis Lactate dehydroganase.

BBa_K4662028: RhlR is an enzyme that acts as a transcription factor, as soon as the concentration of C4-HSL reaches a certain threshold binds and binds to RhlR. In the bound state RhlR can activate LasB transcription of specific genes.

BBa_K4662029: When LasB is activated it will express LDH.

The two level 1 plasmids are connected with two adapters to form a level 2 plasmids.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 714
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 714
  Illegal NheI site found at 29
  Illegal NheI site found at 52
  Illegal NheI site found at 367
  Illegal NheI site found at 608
  Illegal NotI site found at 856
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 714
  Illegal BamHI site found at 708
  Illegal XhoI site found at 865
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 714
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 714
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

LasB dependent expression L-Lactate production by LDH

Figure 1: Measurement of L-lactate abundance in control strains; untransformed and knock out. Comparison to to induced L-LDH via LasB promotor.

To test the efficiency of our L-LDH, we used a L-lactate kit that resulted in luminescence of luciferin, which was measured and used to quantify the amount of L-lactate in the sample. As a negative control we were not only able to use the untransformed bacteria, but also an LDH knockout strain obtained from the KEIO knockout library. We used this strain to express our LDH under a constitutive promoter. Additionally we characterised the lactate production dependent on the LasB promotor. In figure 1 the relative lactate production is illustrated. The LasB promoter successfully activated the LDH expression. Surprisingly, the constitutive promoter did not result in a high production of lactate.

Conclusion

Our rhl-las quorum sensing hybrid system, was successfully adapted for the LasB dependent production of LDH. Thereby we are able to show that by simply exchanging the CFP moiety, our BBa_K4662042 and its property to induce the production of any protein of interest.

Usage and Biology

Part can be used to control the expression of LDH and its capability to acidify the environment.

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.