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Revision as of 01:00, 1 November 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).- 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 potentially 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 Metagenomics and Cloning
Beta-Xylanase
Beta-xylanase was synthesized de novo via our synthetic metagenomic pipeline was cloned into the pET26b(+) expression vector system, under control of the T7 promoter. The construct was then transformed into chemically competent BL21 DE3 E. coli and grown on LB+kanamycin media. After successful transformation, a single colony was grown in batch culture (5 mL LB+kanamycin) until the culture reached an OD of 0.6, at which time 0.1 M IPTG was (or wasn't) added 1:1000 for a final concentration of 0.1 mM. Induction with IPTG continued for 4 hours at which point the bacteria were lysed and proteins collected. Process was repeated for BL21 DE3 E. coli containing empty vector (pET26b+). Proteins were run on a 12% SDS-PAGE gel. After running, the gel was stained with Coomassie Brilliant Blue. A protein around 50 kDa is over-expressed in cells exposed to IPTG with the construct containing beta-xylanase(Fig. 7). Since this protein is not present without induction via IPTG or without the insert and as our protein's predicted molecular weight is ~51 kDa we conclude that this over-expressed protein is our novel beta-xylanase. This assay proves that our protein is able to be expressed via IPTG induction in BL21 DE3 E. coli and is not toxic to the growing bacteria, as we expected.Endoglucanase
In order to assess the enzymatic activity of an endoglucanase isolated from Ruminiclostridium thermocellum by last year’s team, this year’s team designed a media pH change assay centered around the dye Congo Red and the substrate carboxymethylcellulose (CMC). CMC, a cellulose polymer, contains a phosphate group that when cleaved by an enzyme to cellobiose, lowers the pH of the surrounding media. Congo Red is able to detect this pH decrease as a colour-changed halo around the site of pH decrease. We designed a solid media (CMC+Glucose+Congo Red protocolLINK) to support the growth of bacteria containing endoglucanase in the pET26b(+) expression vector. We hypothesized that endoglucanase activity would induce this pH change, showing diffusion of our enzyme and positive enzyme activity. BL21 DE3 E. coli containing endoglucanase (or empty vector alone) were cultured for 12 hours in 5 mL LB Broth+Kanamycin at 37⁰C, shaking. At 12 hours, 0.1M IPTG was added 1:1000 to the culture for a final concentration of 0.1 mM and incubated at 37⁰C, shaking, for another hour. After this hour, culture was streaked onto Congo Red plates and incubated for 24 hours at 37⁰C. Progression was measured at 24, 48, and 72 hours. Both empty vector and endoglucanase containing E. coli were able to produce halos due to pH decrease at the same rate (Fig. 9). As this protocol is traditionally for organisms that can degrade CMC to glucose instead of only degrading CMC to cellobiose, our addition of glucose to the media may have caused this pH change in both samples due to glucose metabolic pathways. In the future, different carbon sources may be useful to prevent this pH change but still allow bacterial growth without the full cellulose degradation pathway (such as xylose).Project Achievements
Over the course of the 2017 iGEM season, we have had some downs, but many more ups. SuccessesNeufeld, J., Engel, K., Cheng, J., Moreno-Hagelsieb, G., Rose, D. and Charles, T. (2011). Open resource metagenomics: a model for sharing metagenomic libraries. Standards in Genomic Sciences, 5(2), pp.203-210.