Part:BBa_K4652002

T7-RBS-SpyTag-GFP-SpyCatcher-Tr

THERMOSTABILITY 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.

THERMOSTABLE CYCLIZED SPYTAG-GPF-SPYCATCHER

Plasmid Construction

To enhance the thermostability of the GFP protein, we deleted start (ATG) codon and stop (TAA) codon, and incorporated SpyTag at the N-terminus and SpyCatcher at the C-terminus. This DNA fragment was synthesized by Integrated DNA Technologies, Inc. (IDT) and then cloned into pSB1C3 (SpyTag-GFP-SpyCatcher, Basic Part:BBa_K4652000). Then, the part was connected with a T7 promoter (Part:BBa_K1833999), a strong RBS (Part:BBa_B0030), and a double terminator (Part:BBa_B0015). This setup mirrored the context of pT7-eGFP (Part:BBa_K1833000), with the exception of the added SpyTag and SpyCatcher. The final construct was verified using colony PCR (Figure 2) and further validated through DNA sequencing. This resultant construct was designated as the improved BioBrick part, pT7-SpyTag-GFP-SpyCatcher (Composite Part:BBa_K4652002).

Figure 2. Verification of pT7-SpyTag-eGFP-SpyCatcher (Part:BBa_K4652002) using colony PCR. PCR was performed using a CmR-specific forward primer from the vector and a SpyCatcher-specific reverse primer from the gene. The expected size of the amplified DNA fragments is 2204 bp. The rightmost lane displays a 1 kb DNA ladder. The numbers correspond to selected colonies, with one control derived from a mock pick from a clear zone on the plate.

Thermostability of SpyTag-GFP-SpyCatcher

90°C treatment

To assess whether the thermostability of GFP was enhanced by the addition of SpyTag and SpyCatcher, lysates from the transformed E. coli BL21, induced with IPTG, were subjected to a heat tolerance test at 90°C – a temperature known to degrade wild-type GFP (as depicted in Figure 1 in CONTRIBUTION). As illustrated in Figure 3, despite a pronounced decline in activity within the first minute of treatment, the fluorescence intensities remained relatively consistent up to 3 minutes. The retention of 22% GFP activity indicates a marked improvement in thermostability compared to the mere 1% observed for wild-type GFP at 90°C after 3 minutes (Figure 4).

Figure 3. Thermostability of cyclized SpyTag-GFPmut3b-SpyCatcher protein at 90°C. E. coli BL21 transformed with BBa_K4652002 was induced using 0.3 mM IPTG at 25°C for 20 hrs. Bacterial lysates were subjected to 90°C treatment for 3 min with an interval of 0.5 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.

Figure 4. 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 at 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]

  Illegal BsaI.rc site found at 759