Team:Toronto/Notebook-w02-thu
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Week 2: May 26, 2016
Thursday, 5/26
Members: Marc, Celine, Karim, Andrea, Bohdan, Kat, Alex, Tam
LAB STUFF:
LacZ alpha complementation:
By Kat Jaenes
For using LacZ - need your E.coli strain to be deleted in LacZ alpha, and then complement with your plasmid (for more info see: https://en.wikipedia.org/wiki/Blue_white_screen)
Relevant genotypes:
DH10B
F– endA1 deoR+ recA1 galE15 galK16 nupG rpsL Δ(lac)X74 φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) StrR λ–
- High competence
- Blue/white selection
DH5α
F– endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK–mK+), λ–
- Lactose nonutilizing
from: http://openwetware.org/wiki/E._coli_genotypes#DH10B_.28Invitrogen.29
Based on this, we should be able to use E.coli K12 DH10B for alpha complementation, meaning that we can use LacZ alpha as a reporter.
Visualization of GolS homolog protein in E.coli:
By Tam Pham
Based on the structure, it is not recommended to insert His-tag at the N-terminus as it will inhibit with its DNA-binding activity significantly. C-terminal His-tag with its linker seems to avoid the current problem but may still be able to prevent dimerization to occur.
The Au(I) binding pocket suggests that the GolS requires dimerization to secure the metal ions. 1 monodimer structure is capable of binding to 2 Au(I) ions. The amino acid residues that are involved in the Au(I) ion is Cys(112), Cys(120) the first monomer and Ser(77) from the second monomer. Ile(122) also appears to be involved in trapping the metal ions; however, there is no charge on Ile, we suspect that it does not play a chelation role.
Plasmid Design:
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Created a folder 'iGEM 2016 Plasmid Parts" that contains all of the relevant sequences and parts that we should need for our project (except for a few promoters and the RBS). Included is the iGEM York 2013 pGolTS/GolS construct from the registry.
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A feature library was created: "iGEM 2016 feature library", and each of the relevant features from the aforementioned folder was added to this library. For future designs that include new features, make sure to add them to this library.
GolS RBS:
Calculated the RBS for GolS using the Salis Lab De Novo Calculator: https://salislab.net/software/forward
The RBS was added to the iGEM 2016 feature library.
- An issue we faced was regarding the pre-sequence for the De Novo DNA:
"Pre-Sequence: enter a nucleotide
sequence (5 to 20 bp) that appears before the ribosome binding site,
using A/G/C/T/U. The Pre-Sequence is important when the ribosome binding
site is less than 35 nucleotides long. (optional)"
Considering that our calculate RBS is 31 nucleotides long, we need to look into designing/inputting a presequence.
Considered ideal promoter among pgolB, pgolTS and pges. The following article was identified as a place to start: http://www.ncbi.nlm.nih.gov/pubmed/17919284.
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In the absence of gold:
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pges: KD=1.4 +/- 0.4x10-6M
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pgolB: KD=5.8 +/- 1.2x10-8M (highest affinity for DNA in the absence of gold!)
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pgolTS: KD=9.9 +/- 2.1x10-8M
ADMINISTRATIVE:
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Had our interview with Dr. Mahadevan, during which period he signed our RRFs.
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Submitted our RRFS for review (all except for Celine, who will be writing her Chem. safety test tomorrow and submitting her RRF afterwards)
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Krishna emphasized the importance of reading SDS before lab, and recieving proper training for Liquid Nitrogen and Gold Chloride
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Recommended looking into Ni biosensing, as a professor nearby works with nickel bioleeching and might be interested in collaboration. They also might have worked with gold chloride. His name is Vladimiros (Vlad) Papangelakis
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Created a notebook entry skeletons for both in lab and out of lab periods
EMAIL UPDATES:
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Consider emailing Dr. Papangelakis
MEETING NOTES:
Shiny Rocks Team:
- Salient points from the presentation by Jake the geologist: [be sure to check out the slides below!]
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Lower the pH, higher concentration of gold
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Biosensor would most likely be used for faults and streams.
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Gold has a high density therefore it is located lower in the ground, making mining expeditions more expensive. Gradient of associated metals: Fe -> Fe-Cu -> Cu-Fe-Zn -> Pt-Zn-Ba-Au
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There are common minerals associated with gold (other sulfide bearing minerals)
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Apparently there's an X-ray method of figuring out the quantity of gold present in a soil sample, however this cannot be done on site
Synenergene:
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Apparently the two directions that synenergene is looking into involve mosquito borne illnesses and biodiversity conservation? They are trying to make their proposal fit into these categories
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Their previous direction: Gold mining -> excess water ie. tailings -> water is a incubator for mosquitoes -> mosquitoes carry malaria
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Adjusted direction: Look into paper based biosensing for the detection of other environmentally influential metals.
FOR TOMORROW:
- Figure out how to design/obtain a pre-sequence for the RBS calculator. Potentially email Kayla about this issue.
- Finally figure out which promoter to use?
- Create a plasmid design from our feature library
- Soon we shall find out the outcome of our CRISPR grant....
