Part:BBa_K4652002

T7-RBS-SpyTag-GFP-SpyCatcher-Tr

THERMOSTABILITY CHARACTERIZATION OF WILD-TYPE GFP

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

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]

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