Part:BBa_K3440004

GFP under Prhl

Prhl(BBa_R0071) - RBS(BBa_B0034) - GFP(BBa_E0040)

Usage and Biology

This part can be used to produce GFP under Prhl, which can be activated by the complex RhlR:C4-HSL. GFP is a fluorescent reporter gene originally found in Aequorea victoria (Jellyfish) (UniProtKB - P42212). It gives a green color (emission at 530nm) when excited at 483nm. pLux promoter is found in Vibrio fischeri (UniProtKB - P12747) and has a role in the quorum sensing system we use. In our project, we rely on this promoter to activate genes when RhlI is expressed upstream. RhlI expression release C4-HSL, a signalling molecule that can be complexed with RhlR on the receiver end and subsequently activates pRhl.

In our project, we use the BBa_K344004 part in order to test the leakiness of pRhl 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_K3440004, we picked colonies from plates (Figure 1) and PCR amplified them with primers VF and VR2.

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  Figure 1: Transformation plate for BBa_K3440004(E)
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  Figure 2: Colony PCR gel for BBa_K3440004(E)and BBa_K3440005(F)

We ran gels of the product at 180V and for 30 mins (Figure 2 and 3). We obtained the expected size for the bands (1111bp) for E10, E5 and E6. We therefore prepared plasmid preparations and glycerol stocks of E10 and sent it for sequencing to Microsynth AG. The sequence obtained corresponded to the expected part for E5.

Figure 3: Colony PCR gel for BBa_K3440004(E5 and E6 successful)

We then proceeded to test the leakiness of pRhl promoter thanks to the GFP reporter added in the part. We measured fluorescence intensity (excitation 483nm, emission 530nm) of GFP for E5,E6 and E10 and calibrated the values with OD600 measurements. The obtained results (Figure 4) show as expected a low expression of GFP under uninduced pRhl (<1000AU).

Figure 4: Fluorescence measurements for BBa_K34440004(E)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 723