Latest revision as of 02:29, 1 September 2023

pT7-eGFP

This part produces GFP in the presence of T7 RNA polymerase. It contains a T7 promoter, a strong RBS, the coding region for GFP, and a double terminator. The GFP is a mutant known as GFPmut3b, and the original citation is as follows:

Cormack, B.P., Valdivia, R.H., and S. Falkow. FACS-optimized mutants of green fluorescent protein (GFP). Gene 173: 33-38 (1996).

(http://www.sciencedirect.com/science/article/pii/0378111995006850)

It has a maximum excitation at 501 nm and maximum emission at 511 nm. For more information, see part BBa_E0040.

Usage and Biology

Figure 1. A typical expression profile of pT7-eGFP in NiCo21(DE3) cells. (Note that error bars are only shown for the negative control and 0.2mM IPTG induction; however, the error bars of the other samples are comparable to the 0.2mM IPTG curve. Each sample was tested with 6 replicates.) IPTG induction of the cells lifts the repression of lacUV5 promoter controlling the expression of T7 RNA Polymerase. The eGFP in this part can then be transcribed and translated. The concentrations of IPTG were chosen based on the recommendation that BL21 (DE3) cells and derivatives be induced with IPTG concentrations between 0.5mM and 1mM. As seen from the graph, during the first two hours, 0.2 mM IPTG induced the cells equally as well as did 1mM IPTG. However, after the first two hours, the cells induced with smaller concentrations of IPTG outgrew the other cultures, which ended up yielding more GFP fluorescence. The OD600s of the cultures at 5 hours are shown below in Figure 2.


Figure 2. The OD600 of the cultures in the experiment from Figure 1 at 5 hours. The difference of OD600s was even more pronounced as the experiment proceeded.

THERMOSTABILITY CHARACTERIZATION by MINGDAO IGEM 2023

The BioBrick pT7-eGFP (Part:BBa_K1833000) comprises a T7 promoter, RBS, and terminator, with an inserted gene of GFPmut3b. This plasmid was retransformed into E. coli BL21. Upon 0.3 mM IPTG induction at 25°C for 20 hrs, bacterial lysates underwent heat tests at varied temperatures for 3 minutes, as indicated in Figure 1. Relative to the untreated control, fluorescence fold change depicted thermostability trends from 70°C, 80°C, to 90°C, retaining 59%, 12%, and 1% of the GFP signal, respectively. This data provided insights into the innate heat tolerance properties of GFPmut3b protein.

Figure 1. Thermostability of GFPmut3b protein. E. coli BL21 transformed with BBa_K1833000 was induced using 0.3 mM IPTG at 25°C for 20 hrs. Bacterial lysates were subjected to temperatures of 70°C, 80°C, and 90°C for 3 min each. Subsequently, the fluorescence of 100μl from each treated lysate was measured at Ex/Em = 483/513 nm. All values were normalized to the average of the untreated control, with the resulting ratio representing the fluorescence fold change.

THERMOSTABLIZING GFP PROTEIN BY CYCLIZATION

To engineer a thermostable protein, using the SpyRing technique can cyclize a protein and make it more heat resistant¹. This involves cloning the protein of interest between the SpyTag at the N-terminus and the SpyCatcher at the C-terminus. We engineered a SpyTag-GFP-SpyCatcher construct (i.e., T7-SpyTag-GFP-SpyCatcher) and evaluated its thermal resistance (Figure 2). See the construction process and characterization detail in Part:BBa_K4652002.

Figure 2. Comparison of wild-type GFPmut3b and cyclized SpyTag-GPTmut3b-SypCatcher proteins. E. coli BL21 transformed with indicated plasmid was induced using 0.3 mM IPTG for 25°C for 20 hrs. Bacterial lysates were subjected to temperatures of 90°C for 3 min. The fluorescence of 100μl lysates was measured at Ex/Em = 483/513 nm. All values were normalized to the average of the untreated control, with the resulting ratio representing the fluorescence fold change.

REFERENCE

1. Schoene C, Bennett SP, Howarth M. SpyRings Declassified: A Blueprint for Using Isopeptide-Mediated Cyclization to Enhance Enzyme Thermal Resilience. Methods Enzymol. 2016;580:149-67. doi: 10.1016/bs.mie.2016.05.004. Epub 2016 Jun 16. PMID: 27586332.

Sequence and Features