Team:Stockholm/9 September 2010

From 2010.igem.org

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==Andreas==
==Andreas==
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Me and Mimmi were discussing the upcoming expression of SOD and its helper chaperone yCCS. Based on an article by [http://www.ncbi.nlm.nih.gov/pubmed/15358352 Ahl, Lindberg and Tibell (2004)], we decided that the two proteins should be expressed in equal amounts (1:1) from the same vector. Since we only have one expression vector (pEX) available, this requires some modifications.<br />
Me and Mimmi were discussing the upcoming expression of SOD and its helper chaperone yCCS. Based on an article by [http://www.ncbi.nlm.nih.gov/pubmed/15358352 Ahl, Lindberg and Tibell (2004)], we decided that the two proteins should be expressed in equal amounts (1:1) from the same vector. Since we only have one expression vector (pEX) available, this requires some modifications.<br />
Our idea is to construct a SOD/yCCS operon from which the two genes can be co-transcribed. This will require a new Shine-Dalgarno (RBS) sequence for translation of the second gene in the operon.
Our idea is to construct a SOD/yCCS operon from which the two genes can be co-transcribed. This will require a new Shine-Dalgarno (RBS) sequence for translation of the second gene in the operon.
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====Extraction of RBS BioBrick (BBa_B0030)====
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Extracted BBa_B0030 (RBS 30), carried on pSB1A2, from iGEM plate 1, well 1H. Transformed into Top10.
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*Quick transformation
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*1 &mu;l DNA
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*Amp 100
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===Cloning of His&sdot;SOD into pMA===
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====Sequencing results====
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*pMA.his.SOD_premix ([[media:PMA.his.SOD_premix_9sep.txt|fasta]])
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Correct sequence verified by Blastn ([[media:Blastn_pMA.his.SOD_premix-pMA.His*SOD_9sep.txt|results]]). Three silent mutations in the His-tag, as has been previously observed.
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== Mimmi ==
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=== SOD.his / his.SOD / yCCS ===
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==== Plasmid prep. ====
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*Follow E.Z.N.A protocol
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**Wash 2x with DNA wash buffer
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**Eluate in 50µl sH<sub>2</sub>O
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{|
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! DNA conc.
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| ng/µl
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|-
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| pSB1C3.SOD.his
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|
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|-
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| pSB1C3.his.SOD
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|
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|-
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| pSB1C3.SOD.his
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|
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|-
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 +
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==== Glycerol stocks ====
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*Add 1800µl to 200µl pre-sterelized glycerol
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=== MITF-M ===
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==== Site-Directed Mutagenesis ====
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{|
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! mix
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| (µl)
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| rowspan="9" width="100" |
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! primers
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| rowspan="9" width="100" |
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! colspan="2" | conditions
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| rowspan="3" |
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|-
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| sH<sub>2</sub>O
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| 40
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| MITF_1F
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! time
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! &deg;C
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|-
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| dNTP
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| 1
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| MITF_1R
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| 2m
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| 95
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|-
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| F primer
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| 1
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| rowspan="6" |
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| 30s
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| 95
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| )
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|-
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| R primer
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| 1
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| 30s
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| 55
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| > 22 cycles
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|-
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| Pfu buffer
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| 5
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| 7m
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| 68
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| )
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|-
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| Pfu turbo
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| 1
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| oo
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| 4
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| rowspan="3" |
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|-
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| DNA
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| 1
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| rowspan="2" |
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|-
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| align="right" | tot
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| 50µl
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|}
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{{Stockholm/Footer}}

Latest revision as of 11:01, 26 October 2010

Home Project Idea Lab Work Results Modelling Team       Follow us on and        2010.igem.org

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Contents

Andreas

Cloning of N-CPPs into pSB1C3

Since we realized that the method we used for cloning the N-CPPs can cause also the intervening sequences to insert into pSB1C3, I decided to redo some clonings. Since the intervening sequences were designed with unique restriction sites, digestion with these endonucleases should prevent cloning of these.

Digestion of N-CPP cluster

[N-CPP plasmid] = 672 ng/μl

Tested the FastDigest buffer, even though conventional Fermentas restriction enzymes were used.

  N-CPP
1st incubation
10X FastDigest buffer 3
DNA (2 μg) 3
dH2O 19
XbaI (conv.) 1
AgeI (conv.) 1
  27 μl
2nd incubation
FD BamHI 1
FD HindIII 1
  29 μl
  • 1st incubation: 37 °C, 2:30
  • 2nd incubation: 37 °C, 0:30
  • Inactivation: 80 °C, 20 min

Ligation

Two ligation reactions were prepared to test the efficiency of two different ligation buffers.

  • Vector: Dig pSB1C3 X+A EXTR (13.72 ng/μl)
  • Insert: Dig N-CPP X+A 9/9 (31.25 ng/μl)
  Lig pSB1C3
NCPP 1
9/9 		Lig pSB1C3
NCPP 2
9/9
5X Rapid Ligation buf. 4 0
10X T4 DNA ligase buf. 0 2
Vector DNA 4 4
Insert DNA 11 11
dH2O 0 2
T4 DNA ligase 1 1
  20 μl 20 μl
  • Incubation: 22 °C, 16 min

Digestion of previous ligation sample

Ligation mix: Lig pSB1C3.N-CPP* 6/9
Ligation mix 15
10X FD buffer 2
FD BamHI 1
FD HindIII 1
  19 μl
  • Incubation: 37 °C, 30 min
  • Inactivation: 80 °C, 15 min

Transformations

Standard transformation protocol.

  • 3 μl ligation mix
    • Lig pSB1C3.N-CPP 1 9/9
    • Lig pSB1C3.N-CPP 2 9/9
    • Lig pSB1C3.N-CPP * 6/9
  • Cm 25 plates

Joint expression of SOD and yCCS from pEX

Me and Mimmi were discussing the upcoming expression of SOD and its helper chaperone yCCS. Based on an article by [http://www.ncbi.nlm.nih.gov/pubmed/15358352 Ahl, Lindberg and Tibell (2004)], we decided that the two proteins should be expressed in equal amounts (1:1) from the same vector. Since we only have one expression vector (pEX) available, this requires some modifications.
Our idea is to construct a SOD/yCCS operon from which the two genes can be co-transcribed. This will require a new Shine-Dalgarno (RBS) sequence for translation of the second gene in the operon.

Extraction of RBS BioBrick (BBa_B0030)

Extracted BBa_B0030 (RBS 30), carried on pSB1A2, from iGEM plate 1, well 1H. Transformed into Top10.

  • Quick transformation
  • 1 μl DNA
  • Amp 100

Cloning of His⋅SOD into pMA

Sequencing results

  • pMA.his.SOD_premix (fasta)

Correct sequence verified by Blastn (results). Three silent mutations in the His-tag, as has been previously observed.




Mimmi

SOD.his / his.SOD / yCCS

Plasmid prep.

  • Follow E.Z.N.A protocol
    • Wash 2x with DNA wash buffer
    • Eluate in 50µl sH2O

Glycerol stocks

  • Add 1800µl to 200µl pre-sterelized glycerol


MITF-M

Site-Directed Mutagenesis

DNA conc. ng/µl
pSB1C3.SOD.his
pSB1C3.his.SOD
pSB1C3.SOD.his
mix (µl) primers conditions
sH2O 40 MITF_1F time °C
dNTP 1 MITF_1R 2m 95
F primer 1 30s 95 )
R primer 1 30s 55 > 22 cycles
Pfu buffer 5 7m 68 )
Pfu turbo 1 oo 4
DNA 1
tot 50µl





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/

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