Part:BBa_K1001757
Lux promoter ahead of GFP
This is a Lux promoter upstream of a GFP
Additions from Team Stockholm 2020
We worked on an identical part (https://parts.igem.org/Part:BBa_K3440003) in order to test the leakiness of the Plux promoter in the context of our project, S-POP. In our final prject, Plux is part of an oscillatory system regulating the expression of mtrb, an electricity production gene in Shewanella oneidensis. Plux is used in combination with quorum sensing molecules in order to control the expression of mtrb. LuxR and AiiA are under control of Prhl, and LuxR activates Plux in combination with AHLs while AiiA degrades the AHLs, thus creating oscillations (see https://2020.igem.org/Team:Stockholm/Model) for a full description.
We created a composite part BBa_K3440003 made of Plux (BBa_R0062) - RBS (BBa_B0034) - GFP(BBa_E0040) which has the same sequence as this part and characterised it (usage, biology and characterization of BBa_K3440003=BBa_K1001757 can be found below) by sequencing and fluorescence intensity measurements.
Usage and Biology
This part can be used to produce GFP under Plux, which can be activated by the complex LuxR:3OC6-HSL. GFP is a fluorescent reporter gene originally found in Aequorea victoria (Jellyfish) [1]. It gives a green color (emission at 530nm) when excited at 483nm. pLux promoter is found in Vibrio Fischeri and has a role in the quorum sensing system we use. In our project, we rely on this promoter to activate genes when LuxI is expressed upstream. LuxI expression release 3OC6-HSl, a signalling molecule that can be complexed with LuxR on the receiver end and subsequently activates pLux.
In our project, we use the BBa_K344003 part in order to test the leakiness of pLux promoter.
Characterization
Due to the pandemics, we haven’t been able to use biobricks to create the iGEM Stockholm 2020 parts. Those parts were ordered as gene blocks from Integrated DNA Technologies Inc.. As a result, the sequences of the biobricks used are the same, but the scars between biobricks might differ, as well as the final size of the part.
After heat shock transformation of the pSB1C3 plasmid containing the BBa_K3440003, we picked colonies from plates (Figure 1) and PCR amplified them with primers VF and VR2. We ran gels of the product at 180V and for 30 mins (Figure 2). We obtained the expected size for the bands (1113bp) for D1.
We therefore prepared plasmid preparations and glycerol stocks of D1 and sent it for sequencing to Microsynth AG. The sequence obtained corresponded to the expected part for D1.
We then proceeded to test the leakiness of pLux promoter thanks to the GFP reporter added in the part. We measured fluorescence intensity (excitation 483nm, emission 530nm) of GFP for D1 and calibrated the values with OD600 measurements. The obtained results (Figure 3) show as expected a low expression of GFP under uninduced pLux (<1000AU).
To conclude, Team Stockholm 2020 added to the characterization of this part by using fluorescence measurements and by sequencing it.
End of additions by Team Stockholm 2020
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 725
References
[1] Uniprot entry P42212
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 725
| control | luxR, HSL |
| direction | Forward |
| negative_regulators | |
| o_h | |
| o_l | |
| positive_regulators | 1 |