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

The emission spectra of all members of the collection were measured in the same plate reader in parallel experiments and the results were normalized by dividing by the OD600 (Figure 1).

Figure 1: Fluorescence spectrum of E. coli BL21 expressing GFP under five different constitutive promoters, at the excitation wavelength of 488 nm.

The emission peak is found at 511 nm, which is in accordance with what was documented by Cormack et al. [1]. In figure 1, the difference of fluorescence intensity between each individual strains is, as expected, related to the promoters strengths.

For comparison, all fluorophore spectra were also recorded in a dilution more suited for their emission intensity and normalized by 1 to the maximum intensity of each construct (Figure 2). From this figure we can conclude that all GFP biobricks function properly and that the emission peak is consistent in at all expression strengths.

Figure 2: Emission spectra of GFP expressed under control of promoters with different strengths, normalized by 1 at maximum intensity. Excitation wavelength was 488 nm.

To investigate the effect of our new BioBricks on cell growth, we performed a growth experiment in which we measure the OD600 and fluorescence intensity over time. Figure 3 shows the 24 hour measurement of optical density and fluorescence intensity. The final OD600 is approximately equal for all different strains, suggesting that the level of constitutive expression was not influencing the growth. Furthermore, fluorescence intensity drops after the exponential growth phase, suggesting that GFP is being broken down by proteases as a response to nutrient limitation. After this event, growth continues at a slower pace, while GFP activity keeps decreasing. All in all, constitutive expression of GFP does not seem to have a detrimental effect on cell growth during exponential phase.

Figure 3: GFP. Kinetic measurement of fluorescence intensity at 522 nm and optical density at 600 nm, while shaking at 37°C. Above 6·10⁴ the intensity was too high to be measured.

Concluding, we successfully improved this part by constructing and characterizing five new BioBricks based on this part. They provide ready-to-go expression devices for green fluorescence, including promoter, RBS, coding sequence and terminators. More information on these parts can be found here: BBa_K1890020, BBa_K1890021, BBa_K1890022, BBa_K1890023, BBa_K1890024.

References

[1] Cormack, B. P., Valdivia, R. H., & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene, 173(1), 33-38.