Part:BBa_K4652010

T7-RBS-SpyTag-PCLase1-SpyCatcher-Tr

Polycaprolactone (PCL) is a biodegradable plastic material that has received FDA approval. It's extensively used in various applications such as tissue engineering, drug delivery, food packaging, and agricultural coverings¹. Typically, products made from PCL take 2-3 years to decompose. As a result, there's a growing interest in identifying novel enzymes that can degrade PCL or in optimizing those that are currently available commercially². However, most of them can’t tolerate at extremely high temperatures, including the boiling temperatures encountered during the thermoforming process of shaping PCL plastic.

In Prof. Fan Li's laboratory, novel PCL-degrading enzymes, PCLase I and PCLase II, were identified and purified from Pseudomonas hydrolytica³. This discovery was made possible by cultivating the bacteria in a PCL-emulsified medium. The team conducted an in-depth study of the PCLase enzymes, examining aspects such as enzyme activity, the influence of pH and temperature, substrate specificity, degradation products, as well as the associated gene sequences and protein structures. Learned with this comprehensive data and the enzymes' impressive PCL-degrading efficiency, our aim is to enhance their thermostability.

SpyRing cyclization technique to enhance enzyme thermal resilience was clarified by Dr. Mark Howarth’s team⁴. SpyRing harbors genetically modified SpyTag (13 amino acids) on the N-terminus and SpyCatcher (12kDa) on the C-terminus on the protein of interest. This context spontaneously reacts together through an irreversible isopeptide bond. SpyRing cyclization was demonstrated successfully to increase stress resilience of β-lactamase and some industrially important enzymes. With this synthetic biology tool, we plan to employ SpyRing cyclization techniques and expect to make PCLase thermal resistent, as demonstrated in our work with the SpyTag-GFP-SpyCatcher construct (Part:BBa_K4652002).

PLASMID CONSTRUCTION

PCLase I and PCLase II gene sequences with N-terminal SpyTag and C-terminal SpyCatcher were synthesized by Integrated DNA Technologies, Inc. (IDT) and then cloned into pSB1C3, respectively (SpyTag-PCLase1-SpyCatcher, Part:BBa_K4652008; SpyTag-PCLase2-SpyCatcher, Part:BBa_K4652012). Then, the parts were connected with a T7 promoter (Part:BBa_K1833999), a strong RBS (Part:BBa_B0030), and a double terminator (Part:BBa_B0015). The final construct was verified using colony PCR (Figure 1) and further validated through DNA sequencing. These resultant constructs were designated as T7-SpyTag-PCLase1-SpyCatcher (Part:BBa_K4652010) and T7-SpyTag-PCLase2-SpyCatcher (Part:BBa_K4652013), respectively.





Figure 1. Verification of T7-SpyTag-PCLase1-SpyCatcher (Part:BBa_K4652010) and T7-SpyTag-PCLase2-SpyCatcher (Part:BBa_K4652013) using colony PCR. PCR was performed using a CmR-specific forward primer from the vector and a PCLase-specific reverse primer from the gene. The expected size of the amplified DNA fragments is 2395 bp for T7-SpyTag-PCLase1-SpyCatcher and 2561 bp for T7-SpyTag-PCLase2-SpyCatcher, respectively. The rightmost lane displays a 1 kb DNA ladder. The numbers correspond to selected colonies, with one control (lane 7) derived from a mock pick from a clear zone on the plate.

REFERENCE

1. Ilyas RA, Zuhri MYM, Norrrahim MNF, Misenan MSM, Jenol MA, Samsudin SA, Nurazzi NM, Asyraf MRM, Supian ABM, Bangar SP, Nadlene R, Sharma S, Omran AAB. Natural Fiber-Reinforced Polycaprolactone Green and Hybrid Biocomposites for Various Advanced Applications. Polymers (Basel). 2022 Jan 3;14(1):182. doi: 10.3390/polym14010182. PMID: 35012203; PMCID: PMC8747341.
2. Urbanek AK, Mirończuk AM, García-Martín A, Saborido A, de la Mata I, Arroyo M. Biochemical properties and biotechnological applications of microbial enzymes involved in the degradation of polyester-type plastics. Biochim Biophys Acta Proteins Proteom. 2020 Feb;1868(2):140315. doi: 10.1016/j.bbapap.2019.140315. Epub 2019 Nov 16. PMID: 31740410.
3. Li L, Lin X, Bao J, Xia H, Li F. Two Extracellular Poly(ε-caprolactone)-Degrading Enzymes From Pseudomonas hydrolytica sp. DSWY01T: Purification, Characterization, and Gene Analysis. Front Bioeng Biotechnol. 2022 Mar 18;10:835847. doi: 10.3389/fbioe.2022.835847. PMID: 35372294; PMCID: PMC8971842.
4. 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
  COMPATIBLE WITH RFC[1000]