Part:BBa_K3484005
Bad promoter + LuxI
This construct synthesizes lactone via the LuxI enzyme. This enzyme is regulated by the pBad promoter, which is induced by arabinose but repressed by glucose. This means that the production of lactone can be controlled in two ways, increasing the production with arabinose and decreasing it with glucose.
A schematic representation of the interactions is shown below (Fig.1). Notice that when LuxI is formed, this enzyme produces lactone (AHL).
This system allows to have a control of the lactone (AHL) production in a really consistent way, and consequently, is a system that could be used to simulate other different ones. The composite part is Biobrick compatible and was characterized in E.Coli Top10.
This composite part is composed by all the following parts:
BBa_B0015:A reliable double terminator that consists on BBa_B0010 and BBa_B0012.
BBa_B0032: A medium RBS. The RBS.3 (medium) (derivative of BBa_0030
BBa_I13453: PBad promoter from I0500 without AraC
BBa_C0061: LuxI gene
Model & Characterization
In order to characterize the pBAD LuxI system we have built a model to compare it to experimental data. It must be taken into account that glucose inhibits the protein production, and arabinose activates it. The following ODE represent the whole system (For full model explanation visit iGEM UPF_Barcelona Model Page).
From the ODE system (Eq.1) we can derive a transfer function if we take into account that LuxI and AHL are on a steady state (dy/dx=0). This transfer function (Eq.2) allows us to connect the concentration of arabinose (ARAB) and glucose (GLU) to the concentration of lactone (AHL) that the cells will produce.
We characterized its behaviour under different concentrations of both arabinose and glucose using BBa_K3484004 as our reporter (We consider its Model and transfer function also for characterizing it, see iGEM UPF_Barcelona Model Page for a detailed explanation). The data was extracted from a Plate-Reader analysis on GFP fluorescence emission with concentrations of Glucose ranging from 100mM to 10uM and concentrations of Arabinose ranging from 1mM to 10nM. This data was fitted using the transfer functions from Equation 2 and the one from BBa_K3484004.
As we can see above (Fig.2), lactone will only be produced in low glucose concentrations and in high arabinose concentrations. Furthermore, we can see that our model follows the experimental data, thus showing us it reflects the real phenomena happening in the producer cells even though some assumptions were made.
Characterization experiments
For the characterization a Plate-Reader analysis was made. All the information on the experimental conditions and parameters used are described on the table below (Table 1).
| Table 1. Plate-Reader Parameters for the characterization of the effects of ASV tag in sfGFP | |||
| Parameters | Value | ||
| Plate-Reader model | Synergy HTX | ||
| Plate type | Thermo Fischer 96-well microplates black-walled clear bottom | ||
| Cell medium | LB | ||
| Time | 24 hours | ||
| Shake | Linear: Continuous, Frequency: 567 rpm (3mm) | ||
| Temperature | 37C | ||
| Gain | 50 | ||
| Optical Density (OD) measurement (absorbance) | 660nm | ||
| GFP excitation wavelength | 485nm | ||
| GFP emission wavelength | 528nm | ||
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 262
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 931
Illegal BamHI site found at 202 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |