Results

Metagenomic Library

DNA Extraction

We were able to confirm the size of the metagenomic DNA isolated from the porcupine fecal samples via pulse field gel electrophoresis (PFGE) (Fig.1). The remainder of the DNA was ran via PFGE and all DNA larger than 24.8 kB was excised without exposure to UV or ethidium bromide. The gel was stained after words for visualization (Fig. 2).

Vector Preparation

pJC8 (Fig. 3) was digested with PmiI and ran on a 0.8% agarose gel. The ladder was stained, and the proper band was marked with a razor. The gel was reassembled and the proper band (11kb) was cut out. The rest of the gel was stained and visualized via UV (Fig. 4). Ligation efficiency of the vector was tested using 3 separated reactions:

1. pJC8 + T4 ligase buffer
2. pJC8+ T4 ligase + ligase buffer
3. pJC8 +T4 ligase + ligase buffer + PNK

These ligation reactions were then transformed into STBL3 E. coli and plated on tetracycline plates.
PmiI is a restriction enzyme that leaves blunt ends, so theoretically pJC8 should be able to re-ligate due to the lack of 5’ phosphates. By adding in PNK, you add the 5’ phosphate groups back allowing for re-ligation. A good result would be low to no colonies on plates 1 & 2, and many colonies on plate 3.
Our transformation plates reflected that we had excellent ligation efficiency with pJC8 (Fig. 5).

Trouble-Shooting

Future Directions of the cosmid library

Sequencing Meagenomic and Cloning

Project Achievements

Over the course of the 2017 iGEM season, we have had some downs, but many more ups.
Successes

• Developed a pipeline to identify, or "mine", the porcupine metagenomic sequencing to discover novel enzymes.
• Identified 8? novel enzymes with variable percent identity.
• Synthesized 5 of those enzymes, and successfully cloned 4 of them into psB1AK3.
• Optimized our previous biobrick Endoglucanse(BBa_K2160000)by adding a C termincal HIS-tag and N terminal PelB sequence (Improve).
• Successfully completed a fluorophore cleavage assay from the Hallam lab.
• Isolated high molecular weight DNA from porcupine fecal samples.
• Obtained efficient ligation and digestion with pJC8 controls.
• Produced phage plaque with the phage packaging extract lamba DNA controls.

Failures

• Did not clone our biobricks into the shipping vector psB1C3
• Was not able to achieve the right environment for our novel beta-xylanase to function
• Was not able to design a functional media assay for our enzymes
• Could not make a functional metagenomic library
• Sheered our high molecular weight DNA