Part:BBa_K1670003

aidA-Promoter + mRFP

BBa_K167003 (P_aidA_mRFP) contains the CepR/I regulated aidA promoter. The CepR binding box is located 44 bp upstream of the predicted transcription start. The part also encodes for a mRFP fluorescent reporter, based on BBa_K1362461. Two silent mutations (T213A, T435A) were introduced to delete two HindIII recognition sites. mRFP can be exchanged using an XhoI restriction site directly upstream of the start codon and the biobrick suffix.

Characterization

BBa_K1670002 (cepR) is characterized along with BBa_K1670003 (P_aidA_mRFP) in a fluorescence-based assay. BBa_K1670002 is cloned into J61002_J23100 (Fig.1) and is afterwards cotransformed together with pSB3C5_BBa_K1670003 (Fig.2) into E.coli BL21.

Overnight cultures are inoculated and diluted to an OD of 0.02 and spotted on microtiter plates. Synthesis of mRFP is induced with different concentrations of octanoyl-homoserine lactone (C8-HSL) ranging from a final concentration of 0.01nM – 100 nM. Cultures are incubated at 37°C in a platereader (Biotek SynergyMX) and fluorescence (Excitation 584 nm, Emission 607 nm) and OD₆₀₀is measured 15 minutes. 10 seconds before measuring the plate is shaked (orbital, medium). As a positive control E.coli BL21 pSB3C5_J04450 diluted to an OD₆₀₀of 0.02 and induced with a final concentration of 0.1 mM IPTG are used. As negative controls and to account for possible auto fluorescence of the cells wild type E.coli BL21 is used. Also sterile controls containing only LB-media without any cells are pipetted on the microtiter plates.

Fig1 . - J61002_BBa_J23100_BBa_ K1670002 (cepR)

Fig 2. - pSB3C5_BBa_ BBa_K1670003 (P_aidA_mRFP)

Results

The constitutively expressed cepR is an activator that will bind to its corresponding promoter P_aidA when a certain C8-HSL concentration is reached, starting the expression of mRFP.

Figure 3 Results of the mRFP emission measurement (607 nm) of E.coli BL 21 J61002_BBa_J23100_BBa_ K1670002 pSB3C5_BBa_K1670003 induced with at C8-HSL concentrations from 0.01 nM to 100 nM, 0 nM and the positive control for mRFP.

As seen in figure 3 the positive control delivered the strongest signal with up to 14900 RFU, followed by the 0.01 nM C8-HSL just above 10000 RFU. The other samples showed a very similar behavior with their emission peaking at around 7000 RFU.
Closer comparing the samples, induced with 0.01 nM, 100 nM, 0 nM respectively and the positive control (Fig. 4) we see that the 0.01 nM sample shows a higher fluorescence emission than the sample without any C8-HSL. However the 100 nM sample had a notably lower emission than the one with 0.01 nM. When cepR binds C8-HSL it should work as an activator for P_aidA. Apparently this is only the case at rather low levels of C8-HSL, like 0.01 nM. At higher concentrations like 100 nM the promoter activity seems to be the same as in the sample without any C8-HSL. It looks like cepR is inhibited in its role as an activator at those C8-HSL concentrations. Considering the fluorescence of the 0 nM sample we can say that P_aidA appears to have a high basal expression without induction by cepR or that cepR is also working as an inducer even when no C8-HSL is present.

Figure 4 Results of the mRFP emission measurement (607 nm) of E.coli BL 21 J61002_BBa_J23100_BBa_ K1670002 pSB3C5_ BBa_K1670003 induced with C8-HSL concentrations of 0.01 nM, 100 nM, 0 nM and the positive control for mRFP. Error bars show the average deviation from the mean of the three measurements.

Another possible explanation for those results could be that at 100 nM C8-HSL is somehow toxic for the cells. Looking at the measured OD₆₀₀ values (Fig. 5) that assumption can be dismissed. All samples carrying the genes for CepR and mRFP grow in a similar manner. After about 10 hours the OD₆₀₀ of the 0.01 nM sample decreases, probably due to nutrient depletion.

Figure 5 Results of the OD₆₀₀ measurement of E.coli BL 21 J61002_BBa_J23100_BBa_ K1670002 pSB3C5_ BBa_K1670003 induced with C8-HSL concentrations of 0.01 nM, 100 nM, 0 nM and the positive control for mRFP. Error bars show the average deviation from the mean of the three measurements.

Results

The constitutively expressed cepR is an activator that will bind to its corresponding promoter P_aidA when a certain C8-HSL concentration is reached, starting the expression of mRFP.

Figure 3 Results of the mRFP emission measurement (607 nm) of E.coli BL 21 J61002_BBa_J23100_BBa_ K1670002 pSB3C5_BBa_K1670003 induced with at C8-HSL concentrations from 0.01 nM to 100 nM, 0 nM and the positive control for mRFP.

As seen in figure 3 the positive control delivered the strongest signal with up to 14900 RFU, followed by the 0.01 nM C8-HSL just above 10000 RFU. The other samples showed a very similar behavior with their emission peaking at around 7000 RFU.
Closer comparing the samples, induced with 0.01 nM, 100 nM, 0 nM respectively and the positive control (Fig. 10) we see that the 0.01 nM sample shows a higher fluorescence emission than the sample without any C8-HSL. However the 100 nM sample had a notably lower emission than the one with 0.01 nM. When cepR binds C8-HSL it should work as an activator for P_aidA. Apparently this is only the case at rather low levels of C8-HSL, like 0.01 nM. At higher concentrations like 100 nM the promoter activity seems to be the same as in the sample without any C8-HSL. It looks like cepR is inhibited in its role as an activator at those C8-HSL concentrations. Considering the fluorescence of the 0 nM sample we can say that P_aidA appears to have a high basal expression without induction by cepR or that cepR is also working as an inducer even when no C8-HSL is present.

Figure 4 Results of the mRFP emission measurement (607 nm) of E.coli BL 21 J61002_BBa_J23100_BBa_ K1670002 pSB3C5_ BBa_K1670003 induced with C8-HSL concentrations of 0.01 nM, 100 nM, 0 nM and the positive control for mRFP. Error bars show the average deviation from the mean of the three measurements.

Another possible explanation for those results could be that at 100 nM C8-HSL is somehow toxic for the cells. Looking at the measured OD₆₀₀ values (Fig. 11) that assumption can be dismissed. All samples carrying the genes for CepR and mRFP grow in a similar manner. After about 10 hours the OD₆₀₀ of the 0.01 nM sample decreases, probably due to nutrient depletion.

Figure 5 Results of the OD₆₀₀ measurement of E.coli BL 21 J61002_BBa_J23100_BBa_ K1670002 pSB3C5_ BBa_K1670003 induced with C8-HSL concentrations of 0.01 nM, 100 nM, 0 nM and the positive control for mRFP. Error bars show the average deviation from the mean of the three measurements.

References

1) Weingart et al (2005) Direct binding of the quorum sensing regulator CepR of Burkholderia cenocepacia to two target promoters in vitro; Molecular Microbiology 57 452-467

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 192
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 752
  Illegal AgeI site found at 864
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 189