Part:BBa_K3786001

GFP expression controlled by Arabinose operon

When transformed into E. Coli, this part allows for the production of GFP only when there is presence of Arabinose and low glucose levels in the medium. The protein of interest needs to be located downstream of the operon to be induced by arabinose. The arabinose operon can be useful to control the production of toxic compounds as this operon allows for a tightly controlled expression of recombinant proteins. The ribosome binding site of this part is the pET RBS (from T7 bacteriophage) which allows for strong expression of protein of interest. This part also contains B0015, which is a double terminator with strong regulation.

Usage and Biology

The pBAD vector system allows for tight and controlled expression of recombinant proteins. Using the Arabinose system for protein production is quite accessible for many reasons:

• L-Arabinose is inexpensive compared to other induction molecules such as IPTG.
• It is very well characterized.
• Using the whole operon to control gene expression leads to a very tight system that can be further controlled by the addition of glucose in the medium.

It’s a system present in E. Coli that allows for the breakdown of L-arabinose. This operon is composed of various genes, however the components of the operon that are most relevant for controlled production of recombinant proteins are the pBAD promoter, pC and araC gene. pBAD is the arabinose promoter and its activation is either regulated or repressed by AraC depending on the presence of L-arabinose in the medium. In the absence of Arabinose, AraC dimerizes to form a loop and binds to the operator regions of pBAD which causes repression of the promoter. When Arabinose is present and glucose is absent in the media, AraC uncleaves this operator region and binds to alternate operator regions of pBAD which will allow its activation, moreover a CAP-cAMP complex forms and binds to pBAD. pC is the promoter activating AraC and both are located on the complementary strand. Low levels of glucose are needed as presence of this sugar in the media leads to low cellular cAMP levels.

In this system, we combined the arabinose operon with a pET ribosome binding site. pET expression vector systems lead to very high levels of protein production, increasing our chances of having a high protein yield

Results and Characterization

After our plasmid was transformed into TOP10 cells that were plated on arabinose plates, the colonies had a very strong green/yellow appearance colour as can be seen on Figure 1.

Figure 1: Yellow colonies from transformation on LB + Kanamycin and Arabinose plates indicating the expression of GFP

For further the characterization of our plasmid, we incubated cells with different arabinose concentrations(20mM - 10mM - 7mM - 5mM - 2mM - 0mM) (Spadiut et al., 2010). M9 medium supplemented with fructose -used as the arabinose system is dependent on the absence of glucose. Cells were then incubated at 37°C for 12 hours with measurements every 10 minutes with Tecan Infinity Pro 200. The settings of the plate reader were determined previously by performing a fluorescein calibration curve.

We can observe in Figure 3 that at the concentration20 mM of arabinose, the OD600 drops after 7 hours of incubation. This indicates that this high concentration of arabinose could affect growth as high concentrations and might lead to cell toxicity. Moreover, what is important to note is that when there was no arabinose added in the culture, almost no fluorescence is observed. This demonstrates that the system is tight and prevents leakiness.

Furthermore, what can be extracted from these graphs is that lower concentrations of arabinose seem to lead to a more stable production of GFP. Indeed, for the cells that were incubated in a medium with a concentration of 2 mM after approximately 5 hours the amount of fluorescence emitted reaches a plateau phase up until the end of the 12 hours although the OD continues to increase. This can also be clearly seen as well for NEB cells grown in 5 mM, 7 mM and 10 mM. There seems to be a point at around 6 hours where fluorescence stops increasing and stays constant although the cells continue growing and dividing. This could be due to the arabinose being almost completely consumed. However, in the case of 20mM arabinose, the fluorescence keeps increasing for a few hours. It would seem arabinose is still available to the cell. Therefore, according to the graphs the optimal L-Arabinose concentration would be between 10 mM and 20 mM.

Further fluorescence characterization studies could be required to find the optimal concentration between the ranges of 10 mM and 20 mM.

Figure 2: Timecourse experiment of the fluorescence normalised to OD600 over 12 hours of NEB Turbo cells transformed with our GFP producing plasmid arabinose inducible and in presence of different arabinose concentrations, as a mean of 3 replicates.

Figure 3: Timecourse experiment measuring the OD 600 of NEB turbo cells transformed with our arabinose inducible GFP producing plasmid in the presence of different arabinose concentrations, as a mean of 3 replices.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1867
  Illegal SapI site found at 961