Experiments

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[CHECK these steps with: TO / AD / LB / DT]

1. Genes, Plasmids, and BioBricks (Genes and Parts that we're working with)
2. Expt 00: Identifying and Confirming Necessary BioBricks from iGEM Parts Registry / Distribution (ID, transform, miniprep (MP), digest, gel)
3. Expt 01: Isolating and Purifying Genes from Lisbon Plasmids (Transform, MP)
4. Expt 02: Confirming Identity of Genes from Lisbon Plasmids (Digest, gel)
5. Expt 03: Converting Lisbon Genes into BioBrick Part (Format) (Primer design/order, (site-directed mutatgenesis) PCR)
6. Expt 04: Confirming Successful Conversion to BioBirck Format (Subclone: digest, ligate, transform, MP, digest, gel)
7. Expt 05: Assembling BioBrick Device(s) (Digest, ligate, transform, MP, digest, gel. Repeat ad infinitum...)
8. Expt 06: Characterisation- Azoreductase Assays (See Standard Operating Procedure (SOP))

Genes, Plasmids, and BioBricks

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No.	ID	Name / Function	Source	State / Concentration / Date Made	Gene Size / Sequence	Initial Plasmid / Vector	Comments
1	pAzoR / BBa_K1336000	FMN-dependent NADH-azoreductase 1	Pseudomonas putida	Miniprep, 48 ng/uL, 15/07/17, TO.	597 bp [Check! Not 612 bp?], Genomic Sequence	Expression vector pET-21a (+) (ampicillin resistant (ampR)), initially cloned between NdeI and BamHI.	Plasmid provided by Lisbon; with Paper.
2	p1B6 (AzoR 1B6) / BBa_K1336001	Mutant: Heat-stable; FMN-dependent NADH-azoreductase 1	Pseudomonas putida	Miniprep, 68 ng/uL, 15/07/14, TO.	597 bp [Check! Not 612 bp?], Genomic Sequence[Add sequence! Sent from Lisbon, see DT]	Expression vector pET-21a (+) (ampR), initially cloned between NdeI and BamHI.	Plasmid provided by Lisbon; with Paper.
3	pCotA / BBa_K1336002	Spore Coat Protein Laccase	Bacillus subtilis	Miniprep, 103 ng/uL, 15/07/14, TO.	1733 bp [Check! Not 1539 bp?] Genomic Sequence[Add sequence! Sent from Lisbon, see DT]	Expression vector pET-21a (+) (ampR), initially cloned between NheI and BamHI.	Plasmid provided by Lisbon; with Paper.
4	LiP / BBa_K1336003	Lignin Peroxidase	Phanerochaete chrysosporium (White-Rot Fungi)	[Being synthesised by Gene Oracle], X ng/uL, dd/mm/yy, Gene Oracle.	X bp, Genomic Sequence	[Cloned directly into expression vector, pSB1C3, between EcoRI and PstI.]	Synthesised (for free) by Gene Oracle. Sequence from Paper.
5	pBsDyP / BBa_K1336004	Dye Decolourising Peroxidase BSU38260	Bacillus subtilis	Miniprep, 51 ng/uL, 15/07/14, TO.	1251 bp, Genomic Sequence[Add sequence! Sent from Lisbon, see DT]	Expression vector pET-21a (+) (ampR), initially cloned between NdeI and BamHI.	Plasmid provided by Lisbon; with Paper.
6	pPpDyP / BBa_K1336005	Dye Decolourising Peroxidase PP_3248	Pseudomonas putida	Miniprep, 55 ng/uL, 15/07/14, TO.	861 bp [Check! Not 864 bp?], Genomic Sequence[Add sequence! Sent from Lisbon, see DT]	Expression vector pET-21a (+) (ampR), initially cloned between NdeI and BamHI.	Plasmid provided by Lisbon; with Paper.
7	BBa_J04450	RFP Coding Device	Spring 2014 BioBrick Distribution. Plate 4, Well 4B. [Check! DT?].	(1) Miniprep, 333 ng/uL, 01/07/14, TO. (2) Miniprep, 38 ng/uL (NanoDrop, dodgy!) 01/07/14, TO.	1069 bp, Genomic Sequence[Add sequence! Made from combined BioBricks?]	Plasmid Backbone: pSB1C3, i.e. chloramphenicol resistant (camR).	LacI-, and CAP-, sensitive; can fail if system contains LacI or CAP protein! RFP Coding Device contains: LacI (R0010), strong RBS (B0034), mRFP1 (E1010), and double terminator (B0015 = B0010+B0012).
8	BBa_R0010	Promoter (LacI regulated)	Spring 2014 BioBrick Distribution. Plate 3, Well 4G. [Check! DT?].	Miniprep, 329.1 ng/uL, 01/07/14, TO.	200 bp, Genomic Sequence[Add sequence!]	Plasmid Backbone: pSB1C3, i.e. chloramphenicol resistant (camR). Plasmid / Vector Map.	This part is an inverting regulator sensitive to LacI and CAP. In the absence of LacI and CAP proteins, this part promotes transcription; in their presence, the part inhibits transcription. LacI can be inhibited by IPTG.
9	BBa_R0011	Promoter (LacI regulated, lambda pL hybrid)	Spring 2014 BioBrick Distribution. Plate 2, Well 6D (Inconsistent sequencing!). [Check! DT? / Maybe use Spring 2013 Distribution, Plate 5, Well 6G.].	Miniprep, 38 ng/uL (NanoDrop, dodgy!), 01/07/14, TO.	55 bp, Genomic Sequence[Add sequence!]	Plasmid Backbone: pSB1C3, i.e. chloramphenicol resistant (camR). Plasmid / Vector Map.	Inverting regulatory region controlled by LacI (BBa_C0010, BBa_C0012, etc.) The PLlac 0-1 promoter is a hybrid regulatory region consisting of the promoter P(L) of phage lambda with the cI binding sites replaced with lacO1.
10	BBa_K314103	Lac induced expression cassette	Spring 2014 BioBrick Distribution. Plate 1, Well 4D. [Check! DT?].	Miniprep, 334 ng/uL, 01/07/14, TO.	1638 bp, Genomic Sequence[Add sequence!]	Plasmid Backbone: pSB1C3, i.e. chloramphenicol resistant (camR). Plasmid / Vector Map.	Lactose (IPTG) inducible protein expression insert includes f1 origin (K314110), a Lac I generator (K314111), a lactose inducible promoter (R0011), and the Elowitz standard RBS (B0034).
11	BBa_K206000	pBAD Strong Promoter	Spring 2014 BioBrick Distribution. Plate 3, Well 14A. [Check! DT?].	Miniprep, 144 ng/uL, 01/07/14, TO.	130 bp, Genomic Sequence[Add sequence!]	Plasmid Backbone: pSB1C3, i.e. chloramphenicol resistant (camR). Plasmid / Vector Map	pBAD is an E. coli promoter that is induced by L-arabinose. In the absence of arabinose, the repressor protein AraC (BBa_I13458) binds to the AraI1 operator site of pBAD and the upstream operator site AraO2, blocking transcription; in its presence, transcription is permitted.
12	BBa_B0034	RBS	Spring 2014 BioBrick Distribution. Plate 4, Well 1N. [Check! DT?].	Miniprep, 156.5 ng/uL, 01/07/14, TO.	12 bp, Genomic Sequence[Add sequence!]	Plasmid Backbone: pSB1A2, i.e. ampicillin resistant (ampR). Plasmid / Vector Map	RBS based on Elowitz (1999) repressilator.
13	BBa_K518012	RBS + RFP + double Terminator	Spring 2014 BioBrick Distribution. Plate 1, Well 18C. [Check! DT?].	(1) Miniprep, 49 ng/uL, 01/07/14, TO. (2) Miniprep, 219.2 ng/uL, 08/08/14, YKH.	828 bp, Genomic Sequence[Add sequence!]	Plasmid Backbone: pSB1C3, i.e. chloramphenicol resistant (camR). Plasmid / Vector Map	This Coding Device contains: RBS.3 (medium) (B0032), mRFP1 (E1010), and double terminator (B0014 = B0012+B0011).
14	[CHECK: BAD PART !] BBa_B0012 (2)	Transcription Terminator for E. coli RNA polymerase	Spring 2014 BioBrick Distribution. Plate 2, Well 2B. [Check! DT?].	Miniprep, 128 ng/uL, 01/07/14, TO.	41 bp, Genomic Sequence[Add sequence!]	Plasmid Backbone: pSB1C3, i.e. chloramphenicol resistant (camR). Plasmid / Vector Map	TE from coliphage T7. This is a bad terminator (Experience: Fails). It is a promoter in the reverse direction.
#	ID	Name / Function	Source	State / Concentration / Date Made	Gene Size / Sequence	Initial Plasmid / Vector	Comments

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Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis.

Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis.