Revision as of 12:40, 9 August 2014

Goodbye Azo Dye : iGEM 2014 - University College London

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About our project

Experiments

Quick Links:

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

Genes, Plasmids, and BioBricks (Genes and Parts that we're working with)
Expt 00: Identifying and Confirming Necessary BioBricks from iGEM Parts Registry / Distribution (ID, transform, miniprep (MP), digest, gel)
Expt 01: Isolating and Purifying Genes from Lisbon Plasmids (Transform, MP)
Expt 02: Confirming Identity of Genes from Lisbon Plasmids (Digest, gel)
Expt 03: Converting Lisbon Genes into BioBrick Part (Format) (Primer design/order, (site-directed mutatgenesis) PCR)
Expt 04: Confirming Successful Conversion to BioBirck Format (Subclone: digest, ligate, transform, MP, digest, gel)
Expt 05: Assembling BioBrick Device(s) (Digest, ligate, transform, MP, digest, gel. Repeat ad infinitum...)
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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@@ Line 30: / Line 30: @@
                  <h1>Experiments</h1>
                  <p><h2>Quick Links:</h2>
-                 Check these steps with: TO / AD / LB / DT
+                 <strong>[CHECK these steps with: TO / AD / LB / DT]</strong>
                  <ol>
-                     <li><a href="/Team:UCL/experiments#Genes">Genes, Plasmids, and BioBricks</a></li>
+                     <li><a href="/Team:UCL/experiments#Genes">Genes, Plasmids, and BioBricks</a> (Genes and Parts that we're working with)</li>
                      <li><a href="/Team:UCL/experiments#Expt00">Expt 00: Identifying and Confirming Necessary BioBricks from iGEM Parts Registry / Distribution</a> (ID, transform, miniprep (MP), digest, gel)</li>
                      <li><a href="/Team:UCL/experiments#Expt01">Expt 01: Isolating and Purifying Genes from Lisbon Plasmids</a> (Transform, MP)</li>
@@ Line 38: / Line 38: @@
                      <li><a href="/Team:UCL/experiments#Expt03">Expt 03: Converting Lisbon Genes into BioBrick Part (Format)</a> (Primer design/order, (site-directed mutatgenesis) PCR)</li>
                      <li><a href="/Team:UCL/experiments#Expt04">Expt 04: Confirming Successful Conversion to BioBirck Format</a> (Subclone: digest, ligate, transform, MP, digest, gel)</li>
-                     <li><a href="/Team:UCL/experiments#Expt05">Expt 05: Assembling a BioBrick Device</a> (Digest, ligate, transform, MP, digest, gel)</li>
+                     <li><a href="/Team:UCL/experiments#Expt05">Expt 05: Assembling BioBrick Device(s)</a> (Digest, ligate, transform, MP, digest, gel. Repeat ad infinitum...)</li>
+                    <li><a href="/Team:UCL/experiments#Expt06">Expt 06: Characterisation- Azoreductase Assays</a> (See <a href="https://docs.google.com/document/d/1sU1CWSoi1Anydh2AkGb7ZnfdKoWgiAIJ7hat0gvctS0/edit?usp=sharing">Standard Operating Procedure</a> (SOP))</li>
                  </ol></p>
                  <h2><a name="Genes">Genes, Plasmids, and BioBricks</a></h2>