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

After 3 failed packaging attempts, we set up our last packaging extract using the control bacteria host E.coli VSC257 and using the lambda phage DNA as the insert in the phage packaging reaction, both supplied by Agilent Gigapack XL III packaging extract.
Unfortunately, no lambda plaques formed at the suggested dilution of 10^-4. This suggests there is an issue with the packaging extract.
After purchasing new packaging extract, a control packaging reaction was set up. This time with the recommended 10^-4 dilution of phage as well as 10^-2 and 10^0 dilutions. Unfortunately, no plaques were seen at the 10^-4 and 10^-2 dilutions, however 10 plaques were counted on the 10^0. This was a slight success; however it is not efficient enough to go forward with our experiment.
Along side trouble-shooting the packaging extract, we ran our insert DNA on a 0.8% agarose gel to ensure that all the high molecular weight DNA was still intact. If intact, we would expect it to stay in the well of the regular gel electrophoresis apparatus. The result of this gel electrophoresis was that all the DNA was sheared down the length of the gel suggesting that no high molecular weight DNA was present (Fig. 6).

Future Directions of the cosmid library

Since our troubleshooting procedures showed that both the packaging extract and the insert DNA was faulty, we have many ways to try and fix the problems in the future.

Packaging Issue
1. The aliquot of packaging extract I used out of the new pack purchased in September had the sample all along the side of the tube. This suggests that the sample may have thawed during transit. As soon as the packaging extract thaws, the constituents of the phage begin to stick together like magnets. It would make sense that efficiency would be much lower in a thawed sample as most of the phage would already have formed capsids, prevent accumulation of foreign DNA and functional phage.
2. Another possibility is the technique used to add the DNA to the packaging extract was imperfect. The Charles lab emphasized that this was a critical aspect of the experiment, so we were sure to take good notes, and look carefully. However it is possible we are missing something and could contact the company for advice if needed.



DNA Issue
1. It is imperative that throughout the process we are handling the DNA with wide-mouth pipet tips. It is probably for that reason that our DNA became so sheared. It is also possible that an accidentally vortex or vigorous shake could have randomly sheared the large and fragile DNA



Other Issues
1. When dephosphorylating the vector pJC8 opt for Shrimp Alkaline Phosphatase instead of Calf Intestinal Phosphatase (CIP). Or if using CIP, do a final phenol extraction before moving forward. CIP is extremely sticky, especially for blunt ended DNA so it’s possible that it hangs on and prevent ligation of pJC8 with the insert DNA from the porcupine microbiome.
2. he regular amount of ATP present in the NEB T4 ligastion kit can inhibit proper blunt-end ligation. In the future, we plan to add our own amount of ATP (5mM) and DTT to Invitrogen’s “ReactOne” buffer which is the exact recipe as the T4 ligase buffer minus the DTT and ATP.




Sequencing Metagenomic 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