Team:Stockholm/9 September 2010

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(Difference between revisions)
(→Andreas)
(→Joint expression of SOD and yCCS from pEX)
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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.
 +
 +
====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 &mu;l DNA
 +
*Amp 100

Revision as of 08:45, 13 September 2010

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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
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