Team:Stockholm/19 August 2010

From 2010.igem.org

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====Assembly and cloning strategies====
====Assembly and cloning strategies====
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[[image:N-cpp_cloning-strategy_19aug.jpg|200px|thumb|right|Cloning strategy for assembly of N-part prefix CPPs (N-CPP) with protein-coding genes and His tag to form ''N-CPP⋅gene⋅His-tag'', into pSB1C3.]]
 +
[[image:C-cpp_cloning-strategy_19aug.jpg|200px|thumb|right|Cloning strategy for assembly of Freiburg prefix CPPs (C-CPP) with protein-coding genes and His tag to form ''His-tag⋅gene⋅C-CPP'', into pSB1C3.]]
Designed cloning strategies for the assembly of '''CPP⋅gene⋅His-tag''' and '''His-tag⋅gene⋅CPP'''; see drawings attached. We will use BioBrick 3A assembly, with vectors carrying the RFP cassette as out destination vector. pSB1A3, pSB1C3 and pSB1K3 are already available with RFP, but not yet pEX.
Designed cloning strategies for the assembly of '''CPP⋅gene⋅His-tag''' and '''His-tag⋅gene⋅CPP'''; see drawings attached. We will use BioBrick 3A assembly, with vectors carrying the RFP cassette as out destination vector. pSB1A3, pSB1C3 and pSB1K3 are already available with RFP, but not yet pEX.
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**pEX
**pEX
-
====Transfer of RFP into pEX====
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===Transfer of RFP into pEX===
-
=====Digestions=====
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====Digestions====
:[pEX] = 161 ng/&mu;l<br />
:[pEX] = 161 ng/&mu;l<br />
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!pSB1C3.RFP
!pSB1C3.RFP
!pEX
!pEX
-
|rowspan="7" valign="middle"|''Incubation: 37 &deg;C, 30 min''
+
|rowspan="7"|''Incubation: 37 &deg;C, 30 min''
|-
|-
|10X FD buffer
|10X FD buffer
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Proceeded to ligation ''without'' enzyme inactivation or DNA purification
Proceeded to ligation ''without'' enzyme inactivation or DNA purification
-
=====Ligation=====
+
====Ligation====
[DNA] = 66.6 ng/&mu;l
[DNA] = 66.6 ng/&mu;l
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|-
|-
|100 ng vector (pEX)
|100 ng vector (pEX)
-
|1.5
+
|width="30"|1.5
-
|rowspan="2"|''1/3<br />ratio''
+
|rowspan="2" width="20"|''1/3<br />ratio''
|rowspan="6"|Incubation: 22 &deg;C, 10 min
|rowspan="6"|Incubation: 22 &deg;C, 10 min
|-
|-
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|}
|}
-
=====Transformation=====
+
====Transformation====
-
Quick transformation as described in protocols. Plated onto 100 Amp LB agar.
+
Transformation according to quick transformation protocol. 3 &mu;l ligation mix. Cells plated onto 100 Amp LB agar.
 +
 
 +
===Assembly of SOD/yCCS&sdot;His into pSB1K3===
 +
(''Step I of N-CPP cloning strategy'')
 +
 
 +
Digestions performed by Mimmi, see section below.<br />
 +
All DNA concentrations in digestion tubes 66.6 ng/&mu;l.
 +
 
 +
====Ligation====
 +
{|border="1" cellpadding="2" cellspacing="0"
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!colspan="4"|Ligation mix
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|-
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|align="center"|[&mu;l]
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!SOD
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!yCCS
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|rowspan="9"|Vector/insert ratio 1:3<br /><br />Incubation: 22 &deg;C, 10 min
 +
|-
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|100 ng vector (pSB1K3)
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|width="30" align="right"|2.2
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|width="30" align="right"|2.2
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|-
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|His insert
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|align="right"|4.9
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|align="right"|4.9
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|-
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|SOD insert
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|align="right"|5.1
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|align="right"|-
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|-
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|yCCS insert
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|align="right"|-
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|align="right"|5.7
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|-
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|5X Rapid Lig. buffer
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|align="right"|4.0
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|align="right"|4.0
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|-
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|dH<sub>2</sub>O
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|align="right"|2.8
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|align="right"|2.2
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|-
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|T4 DNA ligase
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|align="right"|1.0
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|align="right"|1.0
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|-
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|&nbsp;
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|align="right"|'''20'''
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|align="right"|'''20'''
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|}
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 +
====Transformation====
 +
Quick transformation according to protocol. 3 &mu;l ligation mix. Cells plated onto Km 50 LB agar plates.
== Mimmi ==
== Mimmi ==
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:*Add the other restriction enzymes and incubate in 37&deg;C for 30m
:*Add the other restriction enzymes and incubate in 37&deg;C for 30m
:*Inactivate restriction enzymes in 80&deg;C for 5m
:*Inactivate restriction enzymes in 80&deg;C for 5m
 +
 +
----
 +
==Nina==
 +
 +
I worked on obtaining material from the Uppsala iGEM-team and also talked to an Uppsala reseach team in Uppsala University Hospital about artificial skin that they might be able to culture and provide to us for testing the penetration capacity of our future fusion proteins with CPP.
 +
 +
{{Stockholm/Footer}}

Latest revision as of 10:58, 26 October 2010

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Contents

Andreas

Site-directed mutagenesis of SOD & yCCS

Sequencing results

Received the sequencing results for m-SOD 1, m-SOD 2, m-yCCS 1 and m-yCCS 4 from Eurofins. Ran a nucleotide BLAST (BLASTn) alignment to verify successful site-directed mutagenesis.

All sequences revealed an insertion in the Freiburg suffix, between PstI and SpeI sites. We were aware of this, and the insertion will be removed by digestion later.
BLASTn results also revealed three mutations in both yCCS sequences. A protein BLAST (BLASTp) alignment of the translated yCCS sequences revealed all three mutations as silent mutations (BLASTp results).

SOD 1 sample and glycerol stock will be discarded; the other clones are saved as verified.

Assembly of CPP⋅protein⋅His constructs

Brief introduction

Our goal is to assemble our CPPs together with our protein-coding genes and a His tag, in one of two ways:

  • CPPs with N-part prefix (N-CPP): CPP⋅gene⋅His-tag
  • CPPs with Freiburg prefix (C-CPP): His-tag⋅gene⋅CPP

A His-tag (BBa_K157011) is available from the Registry, carried on the pMA (BBa_K157000) plasmid (pMA.His). This has previously been extracted from one of our iGEM plates, and is available both as plasmid, and as transformed cells.
The CPPs have been requested as synthesized DNA genes, and should arrive soon.
At me and Mimmi's lab, yCCS and SOD are now successfully verified in pSB1C3 and ready for cloning.

Assembly and cloning strategies

Cloning strategy for assembly of N-part prefix CPPs (N-CPP) with protein-coding genes and His tag to form N-CPP⋅gene⋅His-tag, into pSB1C3.
Cloning strategy for assembly of Freiburg prefix CPPs (C-CPP) with protein-coding genes and His tag to form His-tag⋅gene⋅C-CPP, into pSB1C3.

Designed cloning strategies for the assembly of CPP⋅gene⋅His-tag and His-tag⋅gene⋅CPP; see drawings attached. We will use BioBrick 3A assembly, with vectors carrying the RFP cassette as out destination vector. pSB1A3, pSB1C3 and pSB1K3 are already available with RFP, but not yet pEX.

Cloning preparations

  • New Km 50 LB agar plates
    • 22 pcs
  • ON cultures (colonies picked from plates stored in 4 °C, grown in 5 ml LB + antibiotic (pMA & pEX in Amp 100; pSB1K3 in Km 50), 37 °C, 250 rpm, ON)
    • pMA.His
    • pSB1K3.BBa_J04450 (pSB1K3.RFP)
    • pEX

Transfer of RFP into pEX

Digestions

[pEX] = 161 ng/μl
[pSB1C3.RFP] = 148 ng/μl
Digestion mix
[μl] pSB1C3.RFP pEX Incubation: 37 °C, 30 min
10X FD buffer 3 3
dH2O 1.5 2.5
2 μg DNA 13.5 12.5
FD XbaI 1 1
FD PstI 1 1
  30 30

Proceeded to ligation without enzyme inactivation or DNA purification

Ligation

[DNA] = 66.6 ng/μl

Ligation mix
100 ng vector (pEX) 1.5 1/3
ratio 		Incubation: 22 °C, 10 min
165 ng insert (RFP) 2.5
5X Rapid Lig. buffer 4
dH2O 11
T4 DNA ligase 1
  20

Transformation

Transformation according to quick transformation protocol. 3 μl ligation mix. Cells plated onto 100 Amp LB agar.

Assembly of SOD/yCCS⋅His into pSB1K3

(Step I of N-CPP cloning strategy)

Digestions performed by Mimmi, see section below.
All DNA concentrations in digestion tubes 66.6 ng/μl.

Ligation

Ligation mix
[μl] SOD yCCS Vector/insert ratio 1:3

Incubation: 22 °C, 10 min
100 ng vector (pSB1K3) 2.2 2.2
His insert 4.9 4.9
SOD insert 5.1 -
yCCS insert - 5.7
5X Rapid Lig. buffer 4.0 4.0
dH2O 2.8 2.2
T4 DNA ligase 1.0 1.0
  20 20

Transformation

Quick transformation according to protocol. 3 μl ligation mix. Cells plated onto Km 50 LB agar plates.

Mimmi

SOD / yCCS

Digestion

pSB1C3.SOD/pSB1C3.yCCS pMA.His pSB1K3.RFP
8<   EcoRI + AgeI 8<   NgoMIV + PstI 8<   EcoRI + PstI
Mix SOD yCCS His-tag RFP
sH2O 7 6 14 11
DNA 19 20 12 15
10X FD buffer 3 3 3 3
EcoRI 0.5 0.5 NgoMIV 0.5 EcoRI 0.5
AgeI 0.5 0.5 PstI 0.5 PstI 0.5
tot 30µl 30µl tot 30µl tot 30µl


  • Incubate with just NgoMIV in 37°C for 1h 30m
  • Add the other restriction enzymes and incubate in 37°C for 30m
  • Inactivate restriction enzymes in 80°C for 5m

Nina

I worked on obtaining material from the Uppsala iGEM-team and also talked to an Uppsala reseach team in Uppsala University Hospital about artificial skin that they might be able to culture and provide to us for testing the penetration capacity of our future fusion proteins with CPP.





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

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