MackenzieRT (Talk | contribs) |
MackenzieRT (Talk | contribs) |
||
Line 265: | Line 265: | ||
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.</br> | 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.</br> | ||
− | Our transformation plates reflected that we had excellent ligation efficiency with pJC8 (Fig. 5). </br> | + | Our transformation plates reflected that we had excellent ligation efficiency with pJC8 (Fig. 5).</br></br> |
<div> | <div> | ||
Line 282: | Line 282: | ||
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). </br></br> | 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). </br></br> | ||
+ | <div> | ||
+ | <center> | ||
+ | <figure> | ||
+ | <img style="background-color:white;" src="https://static.igem.org/mediawiki/2017/b/b1/Fig_6_meta.png" align="middle" width="71%" height"71%"> | ||
+ | <figcaption>Fig6. DNA extracted from porcupine microbiome after blunt-end repair and DNA clean-up.</figcaption> | ||
+ | </figure> | ||
+ | </center> | ||
+ | </div> | ||
<font color= "#C1D35D">Future Directions of the cosmid library</font> </br></br> | <font color= "#C1D35D">Future Directions of the cosmid library</font> </br></br> |
Revision as of 23:31, 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).- 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