Part:BBa_K3440011

GFP under constitutive promoter

Pconst(BBa_J23100) - RBS(BBa_B0034) - GFP(BBa_E0040) - DT(BBa_B0015)

Usage and Biology

This part expresses GFP under constitutive promoter BBa_J23100. 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. In our project, this part was used to test the activity of the constitutive promoter which we have used in several other parts to enable comparison.

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_K3440010, we picked colonies from plates (Figure 1) and PCR amplified them with primers VF and VR2.

Figure 1: plate of N

We ran gels of the product at 180V and for 30 mins (Figure 2). We obtained the expected size for the bands (1226bp) for N1,N2 N3,N4, N6, N7, N11 and N12.

Figure 2: Colony PCR Gel for BBa_K34440011(N), BBa_K3440012(O) and BBa_K3440013(P)

We therefore prepared plasmid preparations and glycerol stocks of those and sent N3 it for sequencing to Microsynth AG. The sequence obtained corresponded to the expected part for N3.

We then proceeded to test the activity of the constitutive promoter thanks to the GFP reporter added in the part. We measured fluorescence intensity (excitation 483nm, emission 530nm) of GFP for N1 and calibrated the values with OD600 measurements. The obtained results (Figure 3 and figure 4) show much lower results than expected.

Figure 3: Fluorescence measurements for BBa_K34440011 (N1,N3,N6)

Figure 4: Fluorescent measurements for BBa_K3440011(N)

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 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 705