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From 2010.igem.org

Contents 1 Hassan 1.1 Folate 1.2 Possible Genes 2 Mimmi 2.1 MITF 2.1.1 - Gel 2.2 MITF + yCCS and RFP 2.2.1 Verification PCR 3 Andreas 3.1 Vector preparation for CPP cloning 3.1.1 Digestion 3.1.2 Gel verification 3.1.3 Gel extraction 3.2 Amp plate preparation 3.3 Cloning of IgG protease 3.4 yCCS clone analysis 3.4.1 Gel verification

Hassan

Folate

below links and articles are meant to be study when I'm finished with interaction map and prove of concept!

• [http://www.ncbi.nlm.nih.gov/pubmed/12761857]
• [http://www.ncbi.nlm.nih.gov/pubmed/17307853,18403248,17698004,17220358,16359333,10201405,15489424,2254281,15865426,17923103,11041843,2185835,11371182,9287033,1958664,8208138,8019142,8408018,8634297,1098865?report=docsum]
• [http://www.ncbi.nlm.nih.gov/pubmed/15611104]
• [http://www.ncbi.nlm.nih.gov/pubmed/17698004]
• [http://www.ncbi.nlm.nih.gov/pubmed/8208138]
• [http://www.genome.jp/kegg-bin/show_pathway?org_name=eco&mapno=00790&mapscale=&show_description=hide]

Possible Genes

• decrease in CAT (Catalase) : [http://www.ncbi.nlm.nih.gov/pubmed/10537016][http://onlinelibrary.wiley.com/doi/10.1034/j.1600-0749.2003.00087.x/abstract]
• increase in SOD (SOD3) : [http://www.ncbi.nlm.nih.gov/pubmed/12692376]
• possible genes involved in Melanocytes adhesion: "An imbalance in cell adhesion molecules was also observed in vitiligo melanocytes. Differentially downregulated genes in vitiligo melanocytes included integrin alpha 7, integrin beta-like 1, integrin beta 7 and catenin alpha-like 1 (cadherin-associated protein). The adhesion between the melanocytes and the basal membrane is mediated by integrins and the contacts between melanocytes and keratinocytes are mediated by cadherins (Hara et al., 1994; Tang et al., 1994; Zambruno et al., 1993). The importance of regulated expression levels of cadherins have also been shown during migration of melanoblasts (Nishimura et al., 1999). It is difficult to predict if this specific downregulation of adhesion molecules impairs the attachment of the melanocytes in the basal layer of the epidermis; however, detachment and transepidermal loss of melanocytes have previously been reported in vitiligo vulgaris (Gauthier et al., 2003)."[http://www.ncbi.nlm.nih.gov/pubmed/18426409] May also look at [http://www.ncbi.nlm.nih.gov/pubmed/17850512] if more interested.

And those from July 22 with data from planning will bring enough explanation for interaction map.

I think by end of this week a wall of text will appear in modelling page!

Mimmi

MITF

- Gel

• No products!

--> Trying more cycles of low annealing temperature

MITF + yCCS and RFP

Verification PCR

Mix	(µl)	X5	X3		yCCS/RFP			MITF
sH2O	22.5	112.5	67.5		pSB1_VF2			MITF_F
F primer	1	5	3		pSB1_VR			MITF_R
R primer	1	5	3		970/925bp			~1300bp
DNA	0.5	4X0.5	2X0.5
tot	25				conditions			conditions
					time	°C		time	°C
					2m	94		2m	94
					30s	94	)	30s	94	)
					30s	60	> 30 cycles	30s	60	> 30 cycles
					2m	72	)	2m40s	72	)
					10m	72		10m	72
					oo	10		oo	10

Andreas

Vector preparation for CPP cloning

pSB1C3 vectors need to be prepared for cloning of our CPPs.

• Digested with NgoMIV (N) & SpeI (S) (for C-CPPs)
• Digested with XbaI (X) & AgeI (A) (for N-CPPs)

Digestion

Vector: pSB1C3.BBa_J18930 A (133.5 ng/μl)

[μl]	N+S	X+A
10X FD buffer	3	3
Vector DNA	25	25
dH2O	0	0
FD XbaI	--	0.5
FD AgeI	--	0.5
FD SpeI	1†	--
NgoMIV (NEB)	1	--
	30	30

† Added after 1:30 of incubation with NgoMIV, since the latter is not a FD enzyme.

Gel verification

1 % agarose, 110 V, 25 min

Expected bands: 2070 bp (pSB1C3), 714 bp (BBa_J18930)

Results
Good digestion, no uncut plasmid. Bands with correct sizes.

Gel extraction

1 % agarose, 70 V, 1 h

DNA concentrations
Sample	Conc. [ng/μl]	A260/A280
pSB1C3 X+A 1	13.72	2.04
pSB1C3 X+A 2	6.397	2.31
pSB1C3 N+S 1	12.22	2.09
pSB1C3 N+S 2	5.807	2.46

Loaded 30 μl of each sample (X+A and N+S). After electrophoresis, 260 mg samples containing digested vectors were excised. DNA purified using the E.Z.N.A. gel extraction kit, following the procedures provided. Each sample eluted twice, as follows:

• pSB1C3 X+A 1: 40 μl
• pSB1C3 X+A 2: 30 μl
• pSB1C3 N+S 1: 40 μl
• pSB1C3 N+S 2: 30 μl

Amp plate preparation

Prepared 20X Amp 100.

Cloning of IgG protease

Re-transformed Top10 with the pSB1C3.IgGp ligation mixture from 3/8 (standard transformation protocol). Plated on Cm 25, incubation ON in 37 °C.

yCCS clone analysis

Ran a gel for Mimmi, where she/we investigated my yCCS clones from 15/7, prior to mutagenesis, comparing them to the RFP (BBa_J18932) gene.

Gel verification

1 % agarose, 100 V, 30 min.

Expected bands:

• yCCS (undig): 1076 bp
• yCCS (dig): 123 bp, 257 bp, 704 bp
• RFP (undig): 1036 bp
• RFP (dig): 1076 bp

Results yCCS B was identified as RFP, which explains the failed yCCS sequencing results. However, yCCS A seems correct, so this clone/sample will be chosen for new site-directed mutagenesis (EcoRI site removal).