Part:BBa_E0840

GFP generator

BBa_E0840 takes as input a transcriptional signal (PoPS) and produce as output the fluorescent protein GFP.

Usage and Biology

• See BBa_E0040 for additional details.
• BBa_E0840 is often used to quantify the behavior of transcriptional control devices such as promoters.
• BBa_E0840 has a strong ribosome binding site.

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 665
  

Improvements

Group: TU Delft 2016

Author: Lycka Kamoen and María Vázquez Vitali

Summary: Part BBa_E0840 does not contain any promoter, thus it can only be used if cloned downstream of one. It is also for this reason that this part (BBa_E0840) did not have any characterization. We have improved this BioBrick in 2 different ways: on the one hand, we provided 5 ready-to-go expression devices for green fluorescence (with promoters of different strengths), which can be used by future teams; on the other hand, this has allowed us to provide characterization for this uncharacterized GFP part. Using PCR primers, we added either of the constitutive promoters BBa_J23100, BBa_J23108, BBa_J23105, BBa_J23117 and BBa_J23113 to this part, to create five new biobrick devices. All of these biobricks were characterized by measuring their spectrum and growth cycle.

New parts: BBa_K1890020, BBa_K1890021, BBa_K1890022, BBa_K1890023, BBa_K1890024.

Characterization

In order to compare this part to the other members of its collection, the spectra were measured in the same plate reader and the results were normalized by dividing by the OD600 (Figure 3).

Figure 3: Fluorescence spectrum of E. coli BL21 expressing GFP under five different constitutive promoters, at the excitation wavelength of 488 nm.

The fluorescence intensity of each of the individual strains is as expected, as compared to the promoter strengths.

For comparison, the emission was normalized by dividing by OD. All strains were measured in the same dilution, in order to make the results reproducible. The emission intensity is as expected, corresponding to the strength of the promoters. All fluorophore spectra were also recorded in a dilution more suited for their emission intensity and normalized by 1 (Figure 3). From this figure we can conclude that all GFP biobricks function properly.

Figure 3, Emission spectra of GFP expressed under control of promoters with different strengths, normalized by 1. Excitation wavelength was 488 nm.