Team:Grenoble-Alpes/Contribution

NeuroDrop

Contribution

CHARACTERIZATION of BBa_J04450

Previous characterizations of BBa_J04450 has shown that the promoter lactose BBa_R0010 has a non-negligible leak and so, express a significant amount of RFP without IPTG induction (Characterisation made by TeamGrenobleAlpes2018). This is the case in most strains of E. coli and can cause issue if you need to have a very controlled protein expression.

Two major factors affect the lactose promoter :

  • IPTG, known to have a positive effect on the transcription of the gene by removing the lac repressor from the DNA.
  • CAP, known to have a positive effect on the transcription when it binds to cAMP by helping the fixation of RNA-polymerase on the DNA.

In standard conditions (without glucose as carbon source) cAMP is synthetized in large amount leading to permanent activation of the cAMP-CAP complex and as a consequence to over-expression of the following gene.

To be able to bind the CAP sites the CAP protein have to first interact with a cAMP molecule. As soon as two cAMP-CAP complexes are bound to the CAP sites the RNA Polymerase initiates transcription.

The goal here is to characterize the effect of cAMP-CAP on the promoter’s leakages. To do that we used the cAMP depleted strain BTH101. In this strain the cya gene has been deleted (cya-) preventing the expression of endogenous adenylate cyclase and so the production of cAMP.

The experiment

Here we transformed BBa_J04450 in:

  1. DH5α which is a standard strain with endogenous adenylate cyclase activity.
  2. BTH101 which is a strain without endogenous adenylate cyclase activity and so without production of cAMP. BTH101 strain is streptomycin resistant.
  3. BTH101 + adenylate cyclase (BTH101-Zip) to generate cAMP in the strain.
    To restore the adenylate cyclase activity we used two plasmids: one containing T18-LeucineZipper and the other containing T25-LeucineZipper. The Leucine Zipper’s will homodimerize and will bring T18 and T25 closer which will restore the adenylate cyclase activity thus allowing the production of cAMP by BTH101. Both T18 and T25 are under the control of an IPTG inducible promoter (lactose promoter). T25-LeucineZipper was cloned in BBa_J04450, and the bacteria was co-transformed with both plasmid T25-LeucineZipper + BBa_J04450 and T18-LeucineZipper.

DH5α, BTH101 (without cAMP) and BTH101-Zip (with cAMP) were tested as follow:

A range of IPTG concentrations and several times of induction were tested. Relative Fluorescence Units (RFU) where recorded with an excitation wavelength of 561nm and emission wavelength of 588nm. The assay was performed in triplicate in a 96 wells NUNC plate.

Induction time Untransformed Strain O mM IPTG O.1 mM IPTG O.3 mM IPTG O.5 mM IPTG O.8 mM IPTG 1 mM IPTG
1h X3 X3 X3 X3 X3 X3 X3
2h X3 X3 X3 X3 X3 X3 X3
3h X3 X3 X3 X3 X3 X3 X3

Results

DH5α transformed with BBa_J04450 and grown overnight on LB-Agar + chloramphenicol.

BTH101 transformed with BBa_J04450 and grown overnight on LB-Agar + Streptomycin and chloramphenicol.

BTH101-Zip with T25-LeucineZipper + BBa_J04450 and T18-LeucineZipper and grown overnight on LB-Agar + Streptomycin and chloramphenicol.

It is already apparent that without IPTG there is a visible difference between strains that can produce cAMP (DH5α and BTH101-Zip) when compared with the BTH101 strain that do not produce any cAMP.

The DH5α red colonies reveal a substantial RFP expression as the result of the leakage of the promoter.

BTH101 has white colonies indicating that no RFP was produced. This data suggest that the promoter does not leak in that condition.

BTH101-Zip presents same red colonies than the DH5 strain.

DH5α

Table 1: Means of triplicate with standard derivation (σ) for each condition. Excitation 561nm and RFU was read at 588nm.

Blank : Untransformed DH5α O mM IPTG O.1 mM IPTG O.3 mM IPTG O.5 mM IPTG O.8 mM IPTG 1 mM IPTG
1h O.31 (σ=0.02) 5.50 (σ=0.37) 9.90 (σ=0.27) 9.51 (σ=0.09) 11.51 (σ=0.09) 10.37 (σ=0.55) 11.57 (σ=0.91)
2h O.30 (σ=0.01) 7.30 (σ=0.36) 9.98 (σ=0.34) 9.61 (σ=0.62) 11.61 (σ=0.96) 12.65 (σ=0.92) 12.98 (σ=0.49)
24h O.32 (σ=0.01) 10.12 (σ=0.56) 10.41 (σ=0.55) 10.67 (σ=0.76) 11.67 (σ=0.52) 14.40 (σ=1.02) 20.40 (σ=0.39)

In a strain producing endogenic cAMP the lactose promoter has a non-negligible leak which triggers RFP expression even without any IPTG induction. The amount of RFP increases over time and IPTG concentration. Furthermore, the longer the induction time the greater the effect of IPTG seems to be.

BTH101 without cAMP

Table 2: Means of triplicate for each condition. Excitation 561nm and RFU was read at 588nm.

Blank : Untransformed BTH101 O mM IPTG O.1 mM IPTG O.3 mM IPTG O.5 mM IPTG O.8 mM IPTG 1 mM IPTG
1h O.31 (σ=0.02) 0.34 (σ=0.02) 0.32 (σ=0.01) 0.43 (σ=0.05) 0.44 (σ=0.04) 0.44 (σ=0.05) 0.44 (σ=0.02)
2h O.30 (σ=0.01) 7.30 (σ=0.36) 9.98 (σ=0.34) 9.61 (σ=0.62) 11.61 (σ=0.96) 12.65 (σ=0.92) 12.98 (σ=0.49)
24h O.32 (σ=0.01) 10.12 (σ=0.56) 10.41 (σ=0.55) 10.67 (σ=0.76) 11.67 (σ=0.52) 14.40 (σ=1.02) 20.40 (σ=0.39)

Strain without endogenous cAMP synthesis has a basal expression lower than the strain that can produce endogenic cAMP.

Neither time nor IPTG have any effect on the RFP expression probably because no lactose promoter activation occurs.

The data suggest that cAMP is essential for the transcription of genes located downstream the lac promoter unlike IPTG.

BTH101 with cAMP

Table 3: Means of triplicate for each condition. Excitation 561nm and RFU was read at 588nm.

Blank : Untransformed BTH101-Zip O mM IPTG O.1 mM IPTG O.3 mM IPTG O.5 mM IPTG O.8 mM IPTG 1 mM IPTG
1h O.30 (σ=0.01) 0.35 (σ=0.03) 7.16 (σ=0.21) 8.42 (σ=0.30) 7.23 (σ=0.23) 7.02 (σ=0.12) 7.27 (σ=0.33)
2h O.32 (σ=0.02) 0.55 (σ=0.09) 9.75 (σ=0.47) 9.63 (σ=0.44) 10.65 (σ=0.61) 10.20 (σ=0.65) 10.97 (σ=0.84)
24h O.31 (σ=0.02) 1.01 (σ=0.08) 10.26 (σ=0.78) 11.16 (σ=0.88) 11.63 (σ=0.82) 12.04 (σ=1.79) 78.71 (σ=1.23)

When the adenylate cyclase activity is restored, cAMP is produced as shown in the BTH101-Zip strain. The level of expression of RFP is comparable to that obtained in DH5α strain indicating that cAMP is an essential factor for transcription under the control of the lactose promoter.

The endogenic and uncontrolled production of cAMP is the cause of the leakage. As a consequence, making the cAMP production dependent on the presence of IPTG the leakage is greatly reduce in this strain.

Because T18-Zip and T25-Zip are under an IPTG inducible promoter they express very low level of RFP in absence of IPTG (very few T18 and T25 are expressed and so very few cAMP are produced).

Conclusion

RFP expression by BTH101 with or without cAMP shows that without cAMP, the lactose promoter has almost no leakage suggesting that the leakage of the lactose promoter is due to cAMP in absence of IPTG. When the cAMP production is restored the promoter is activated and the protein is expressed. It is also interesting to note that with the BT101-Zip there are very little protein expression if no IPTG is added.

Using cAMP depleted strains and T18-leucineZipper/T25-leucineZipper under the control of a lactose promoter is a good alternative to produce inducible proteins without leakage

Leaks without IPTG Protein expression with IPTG Impact of time and IPTG concentration on protein expression
DH5α + ++ +
BTH101 - - -
BH101-Zip - + ++