Part:BBa_K1831000

IceFinity circularization of Type III AFP + Linker

NpuDnaE intein circularization construct using Type III AFP and a computer-optimized linker

What: Starting from the NpuDnaE intein RFC [105] circularization construct BioBrick from Team Heidelberg 2014 (BBa_K1362000 [1]) we developed a computationally-optimized linker sequence to circularize a Type III antifreeze protein. Our linker consists of the tripeptide GAA, which in addition to the extein scar CWE/RGK, links the N and C termini of a the Type III AFP (PDB file: 1AME) without straining the core protein structure or distorting the residues critical for ice-binding. Our BioBrick also added a constitutive T7 promoter (Part:BBa_I14018 [2]) allowing expression of your circular protein without any additional sub-cloning steps.

Why: Type III AFPs have a variety of potential applications in the oil and gas, food, and health care industries, however these industries often use processes involving extreme temperatures, pH levels, and salt concentrations. Type III AFP is a particularly fragile protein as it denatures at 37oC. Circularizing the protein using our BioBrick affords greater stability at a wider variety of experimental conditions.

The cloning of our circAFP began with a Golden Gate Assembly Reaction, the insert being our AFP-linker-extein sequence described above. Golden Gate Assembly involved a one-pot reaction, where both insert and vector were cut then ligated to generate a pSB1C3 plasmid containing our gene of interest. The Golden Gate Assembly Reaction was then transformed onto Topten electro-competent E. coli cells and plated onto Chloramphenicol-resistant plates. Successful cloning was determined by colony growth and colour (Heidelberg’s part contained an RFP; therefore, non-red colonies would indicate successful gene insertion). Colonies were picked and screened for our gene of interest. Next, our gene of interest was then inserted into a plasmid containing a T7 promoter and ribosomal-binding site to be used for protein expression. To determine successful insertion, further colony screening was performed (Figure 1).

Figure 1. Agarose gel depicting screened colonies containing AFP-linker-extein within our vector containing a T7 promoter. Specifically, colonies 1 and 3-6 all appear to contain an insert of the correct size (about 800bp). Those select colonies were sequenced then used for subsequent transformation and protein expression.

Next steps were to attempt ice-affinity purification(IAP) of the circularized AFP, as shown in Figure 2. IAP uses the knowledge that AFPs and other ice-binding proteins attach themselves to ice, which creates an effective method of isolating these proteins from other cell compounds. IAP also verifies that our circAFP is in fact active despite our modifications.

Figure 2. SDS-PAGE of ice-affinity purified circAFP. This gel illustrate the results of one round of IAP. The single band in the last lane indicates the purified circAFP, isolated in the ice fraction. The liquid fraction represents all compounds not incorporated into the ice.

circAFP Thermostability

To test circAFP’s thermostability, we treated three samples at three temperatures 37, 68, and 90 ^oC for 10 minutes each. Wild type samples were also treated at these same conditions. After treatment, TH assays were performed and TH gaps were compared based on percent retention of activity, relative to the untreated sample. Figure 3 shows a bar graph which illustrates these preliminary results.

Figure 3. Bar graph of TH assay results, comparing wtAFP with our circAFP at various activity test conditions. Data shows that circAFP retains almost 80% of its antifreeze activity after being subjected to 90 ^oC . The wild-type AFP quickly loses its activity after exposure to such high temperatures.

NpuDnaE intein for circularization of Type lll AFP+ inker

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]