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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).</br> | 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).</br> | ||
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− | <img src="https://static.igem.org/mediawiki/2017/8/82/Figure_1_meta.png | + | <img src="https://static.igem.org/mediawiki/2017/8/82/Figure_1_meta.png"> |
<figcaption>Fig1. DNA extracted from the porcupine microbiome.</figcaption> | <figcaption>Fig1. DNA extracted from the porcupine microbiome.</figcaption> | ||
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Revision as of 22:51, 31 October 2017
Results
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:- pJC8 + T4 ligase buffer
- pJC8+ T4 ligase + ligase buffer
- pJC8 +T4 ligase + ligase buffer + PNK
- 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.
- 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.
- 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
- 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.
- 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.
- 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.
- 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
- Packaging Issue
- DNA Issue
- Other Issues
Sequencing Metagenomic and Cloning
Project Achievements
Over the course of the 2017 iGEM season, we have had some downs, but many more ups. Successes