Team:Stanford-Brown/SB16 Notebooks UV
Melanin/Radiation
Made with Benchling
Project: iGEM 2016
Authors: Michael Becich, Elias Robinson, Taylor Sihavong
Dates: 2016-06-28 to 2016-10-03
Tuesday, 6/28
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Cultured cells from former SB-iGEM team's plate with melanin-producing cells (Andy Weir-signed plate)
Wednesday, 6/29
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Cells were dead -- need to pivot and find new way to produce melanin
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Julia's old lab has melanocytes that overexpress melanin
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Copper and tyrosine supplemented to growth media boosted pigmentation of melanin
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Cambridge team from registry has version of MelA Tyrosinase necessary (Eumelanin superior to pheomelanin and neuromelanin)
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Coating with Titanium Dioxide to produce a solar cell still an idea down the road
Tuesday, 7/12
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Ordered mutMelA gene (tyrosinase) for synthesis from IDT based on Biobrick BBa_K274001 from iGEM Team Cambridge 2009 that was not in stock at the registry
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This biobrick already contained one substituted nucleotide (C to T at the 1000th nucleotide) resulting in a Pro334 to Ser mutation that was shown to decrease time to onset of melanin production (Santos et al. 2008)
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Changed Asp535 to Gly by substituting a single nucleotide --> this mutation was also shown in a more recent paper to shorten the time to onset of melanin production in E. coli (Chávez-Béjar et al. 2013)
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Began designing primers to insert mutMelA into pSB1C3 backbone with appropriate promoters and RBS
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Continued research on small melanin binding proteins
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Many short polypeptides shown to be effective in binding melanin, linked to a radioactive subunit and tested in vitro and in vivo (in mice) for human melanoma cell localization and radiation delivery
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One decapeptide discovered through the use of a phage display library called 4B4 shown to be effective at binding melanin (Ballard et al. 2011)
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Many phage display library derived heptapeptides shown to be effective (Howell et al. 2007)
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Discussed possibility of designing CBD-Melanin-Binding Peptide connector
ELIAS'S LAB NOTEBOOK
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Ordered MelA gene from iGEM registry
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Next: design plasmid with inducible promoter
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Next: Order primers for linearisation and Gibson
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Melanin binding peptides:
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4B4: Fungal melanin-binding decapeptide
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Sequence: YERKFWHGRH
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Designed oligonucleotide sequences for repetetive sequence assembly (Kosuke's method)
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Planning on assembling as follows:
Screen Shot 2016-08-19 at 9.43.32 am.png
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Should also order & assemble irrelevant decapeptide PA1 as control
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Sequence: LQYTPSWMLV
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Set of heptapeptides: (Howel 2007)
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Phage 4D had highest melanised/non-melanised absorbance ratio, expressed peptide
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His-Thr-Thr-His-His-Arg-Asn
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HTTHHRN
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Phage 3D: Asn-Pro-Asn-Trp-Gly-Pro-Arg (NPNWGPR)
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Phage 1C: Thr-Thr-His-Gln-Phe-Pro-Phe (TTHQFPF)
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Irrelevant control: Asp-Gly-Ala-Trp-Met-Gly-Ala (DGAWMGA)
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Optimising melanin production in E. coli
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Cultivation media & growth conditions
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Melanin producing cell media
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50ml per culture of...
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M9 minimal salts medium
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Supplemented with 2 or 10 g/L glucose
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0.1 mM IPTG
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Required antibiotic (strain-specific)
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0.4 g/L of L-tyrosine
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20 ug/mL
Wednesday, 7/13
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Decided to engineer a repetitive motif of the melanin binding decapeptide 4B4 as well as the most effective melanin binding heptapeptide documented in Howell et al. 2007 (will be refered to as 4D based on cell strain in paper).
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Based on the sizes of the genes that would code for the two candidate polypeptides (30mer and 21mer), we decided to use the methods detailed in Fujishima et al 2015 for overhang based randomized DNA block assembly
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Decided to attach melanin binding repetitive motif to cellulose binding domain
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Plan on demonstrating proof of concept for all in one melanin binding/membrane binding linker to be used as a mechanism for even and resiliant melanin coating to increase UV radiation resistance
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Constructed 45mer GS linker in silico to separate and preserve the functions of the CBD and the melanin binding domain
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Designed single stranded oligonucleotides for DNA building blocks of 4B4 and 4D, as well as non-melanin binding control decapeptide PA1 and heptapeptide P601G from Ballard et al. 2011 and Howell et al. 2007 respectively
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Designed full plasmid construct for melanin binding repetitive motif connected at its C-terminus by a 45mer GS linker to the cellulose binding domain in Biobrick BBa_K1321357, which is followed by a FLAG-lumio-HIS tag for purification, and finally a double terminator
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Decided to linearize pSB1C3 backbone from meffRed BBa_K1033920, including BBa_J23110 constitutive promoter, Elowitz Represilator RBS BBa_B0034, and meffRed start codon since no start codon was included in repetitive melanin binding DNA block
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Began designing primers for linearization of plasmid backbone and PCR amplification of biobricked CBD
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Used SnapGene Gibson Assembly software for primer design
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Included primer tail extensions to create identical overlaps with fragment inserts for later Gibson assembly
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Ran into issues with inconsistent Tm values due to high GC content, as well as secondary annealing sites due to redundancies in pSB1C3 prefix and suffix
ELIAS'S LAB NOTEBOOK
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Assembly of Kosuke fragments:
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Needed enzymes:
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T4 DNA ligase (NEB)
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Cat #: M0202S (400,000 units/ml)
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Cat #: M0202T (2,000,000 units/ml)
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Size: 20,000 units
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Cost: $64
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T4 polynucleotide kinase (NEB)
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Cat #: M0201S (500 units), $56
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Cat #: M0201L (2,500 units), $224
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Concentration: 10,000 units/ml (price: $224)
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ELIM sequencing preparation:
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Premix recipe:
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Total volume: 15ul (in water)
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Template quantity:
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PCR products:
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100-200bp
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200-500bp --> 2-3ng
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500-1000bp --> 6-8ng
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1000-2000bp --> 10-15ng
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>=2000bp --> 60-100ng
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Single stranded: 100-150ng
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Double stranded (plasmids): 500ng
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Primer: 8pmol
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Upregulate carbon flow to aromatic/L-tyrosine
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Got cells with genomic deletions for shuttling carbon into L-tyrosine/aromatics pathway
Screen Shot 2016-08-19 at 10.07.59 am.png
Thursday, 7/14
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Obtained melanocytes from Julia's former lab at VA Hospital and lysed in water to obtain melanin raw source
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Ordered ten single stranded oligonucleotides from ELIM
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Top and bottom strands for 4B4, 4D, PA1, P601G, and GS linker
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Designed so that once annealed, each double stranded oligo would have a six nucleotide double Gly overhang for assembly and ligation
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Continued designing PCR primers for linearization of intended backbone and amplification of CBD for Gibson assembly
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Secondary annealing site issues solved by using NEBuilder online software for primer design: created longer primary annealing regions on primers and increased initial Tm so that secondary annealing would be outweighed by correct annealing of primers
ELIAS'S NOTEBOOK
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Oligos arrived (not linker)
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Fri 7/23: anneal oligos
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Mon 7/26: Phosphorylate & ligate
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Tues 7/27: run gel and extract correct band
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DNA annealing:
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Template:
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Oligo forward (200uM) - 10ul
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Oligo reverse (200uM) - 10ul
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10x H Buffer - 2ul
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DI H2O - 28ul
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Total - 50ul
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Final oligo concentration: 80mM
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Mixed 4B4, 4D, PA1, P6016 in above formula
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Ran in thermal cycler on "anneal" in Kosuke's folder
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Let sit at 4˚C for 15 min then put in -20˚C
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All oligos in orange iGEM 2016 box in -20˚C
Screen Shot 2016-08-19 at 10.13.20 am.png
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Kosuke's paper: "an overhand-based ... random library" (2015)
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Paper suggests annealing of oligos in thermal cycler by decreasing 10˚C every 5 minutes (we used 1-2 minutes)
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Phosphorylation: used T4 kinase in 10x ligase buffer at 37˚C for 1h
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Ligation: T4 DNA ligase added directly to above mix, incubated at room temperature for 2h
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Adapter (blunt ends): phosphorylated, then mixed with phosphorylated DNA blocks in 1:8:1 ratio in 1x T4 DNA ligase buffer (5' adapter : DNA block : 3' adapter)
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Can also incubate at 16˚C overnight to increase efficiency
Friday, 7/15
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Eight of the oligos arrived (everything but the GS linker)
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Annealed oligos using the "Annealing of Template DNA" protocol
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Stored in -20°C fridge for repetitive assembly upon arrival of GS linker
Saturday, 7/16
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Prepared and assembled buffers and enzymes for overnight phosphorylation and ligation of repetitive DNA building blocks following "Phosphorylation and Ligation of Repetitive DNA Building Blocks"
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Used T4 Polynucleotide Kinase and T4 DNA Ligase
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Awaiting arrival of blunt ended GS linker for construction of repetitive motif
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No oligos came, will wait until Monday for reaction
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Monday, 7/18