Part:BBa_K5090009

GFP Device Regulated By lacO and Kink Turn

Description

Green fluorescent protein (GFP) is a protein that originated from the jellyfish Aequorea victoria, capable of emitting photons of light for bioluminescence. (Tsien R, 1998) The protein emits green light (509 nm) upon excitation with ultraviolet (UV) or blue light (at 396 nm or 475 nm) (Remington, 2011).

This part is a functional composite part including an Anderson promoter (BBa_J23100), two lac operators (BBa_K079017), a kink turn (BBa_K4140015), an Anderson RBS (BBa_J61100), GFP (BBa_K5090003), and a T1 terminator (BBa_B0010).

Usage and Biology

GFP is widely used as a reporter gene for monitoring gene expression in vivo assays as it is non-hazardous and is compatible with a variety of organisms including bacteria and mammalian cell lines. In addition, it does not require the addition of substrates, which can sometimes ramp up cost (Chalfie et al., 1994). Many variants of GFP have been developed, which include sfGFP, EGFP, and cGFP to tailor and fine tune for various purposes (Chalfie et al., 1994). Wild-type GFP is the dimmest version out of them all (Zimmer 2002). We think that it could potentially be useful in assays that need to minimize autofluorescence or interference with other experiments.

We built upon the advancements of Laird et al. (2017) and Wang et al. (2023) by implementing the WT GFP gene with two lac operators (BBa_K079017) and a kt kink for the reporter gene (BBa_K4140015) in a bacterial context, as opposed to their mammalian implementation. In this context, the gene is placed under the control of their dual repressors: enhanced LacI (BBa_K5090001), which was developed by Laird et al., and L7Ae (BBa_K5090002).

Characterization

Wet Lab

In Wet Lab, we were able to confirm that WT GFP was successfully cloned into E. coli. Our sequencing results demonstrate that this cloning occurred without any mutations.

Figure 1: Plasmid map showing successful cloning of GFP into pACYC backbone.

Figure 2 :Fluorescence microscopy of MRE600 cells not expressing WT GFP and expressing WT GFP illustrates a difference in fluorescence level that is detectable.

Figure 3: Expression of WT GFP in MRE 600 E.coli shows an increase in fluorescence (Group B) after incubation overnight compared to control.

Figure 4: Expression of WT GFP in cell-free systems shows an increase in fluorescence after incubation for four and six hours compared to control.

Dry Lab

In Dry Lab, we characterized the expression of GFP, and the repression of GFP achieved by the enhanced LacI, courtesy of Laird et al., and L7Ae. Enhanced LacI and L7Ae repressed GFP by different amounts. With these two transcriptional and translational regulators both repressing the same GFP, expression was further reduced than either achieved alone. This demonstrates that this dual-regulation system is capable of significantly reducing expression of genes which have lacO sites and kink turns in advance of their coding sequence, and more so than either repressor alone. For more information, refer to the 2024 Stony Brook iGEM Team’s modeling page.

Figures 5, 6, 7, and 8: Graphs illustrating the effect of the dual repression system upon GFP expression given GFP is placed under the dual-regulation system, as well as that system’s own regulation by miRNA-326 and Ago2.

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
  COMPATIBLE WITH RFC[1000]

References

• Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., & Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science, 263(5148), 802–805. https://doi.org/10.1126/science.8303295

• Lee, K.-H., Oghamian, S., Park, J.-A., Kang, L., & Laird, P. W. (2017). The REMOTE-control system: A system for reversible and tunable control of endogenous gene expression in mice. Nucleic Acids Research, 45(21), 12256–12269. https://doi.org/10.1093/nar/gkx829

• Remington, S. J. (2011). Green fluorescent protein: A perspective. Protein Science: A Publication of the Protein Society, 20(9), 1509–1519. https://doi.org/10.1002/pro.684

• Shu, W.-J., Lee, K., Ma, Z., Tian, X., Jong Seung Kim, & Wang, F. (2023). A dual-regulation inducible switch system for microRNA detection and cell type-specific gene activation. Theranostics, 13(8), 2552–2561. https://doi.org/10.7150/thno.84111

• Tsien, R. Y. (1998). The green fluorescent protein. Annual Review of Biochemistry, 67, 509–544. https://doi.org/10.1146/annurev.biochem.67.1.509

• Zimmer, M. (2002). Green fluorescent protein (GFP): Applications, structure, and related photophysical behavior. Chemical Reviews, 102(3), 759–782. https://doi.org/10.1021/cr010142r