Team:Groningen/Notebook/21 September 2009

From 2009.igem.org

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Wet

GVP Cluster

Planning

TODO work out the wiki page for GVP
  • made a layout
  • still have to read all articles
  • modeling will stay as it is, has been done by modeling people
DONE make a doodle for presentation planning (1-19 oct.)
DONE media attention
  • mail to UK, Ing., St. Gen.
  • facebook account with link to twitter
  • ethics survey link on facebook and twitter
DONE place an ethics survey link on twitter


DONE clone pArsR-GVP into pSB2K3
  • TODO still need to make glycerol stock
TODO clone repeat out of GVP cluster
TODO make glycerol stocks of constructs
TODO enter info on part registry


Restriction for Assembly

The plasmid of pMA-gvpL insert was cut with MvaI/XhoI to cut out the entire part of wanted fragment.

Plasmid Amount μL MQ μL R-digest buffer MvaI digest enzyme XhoI digest enzyme
pArsR-GVP no.1 10.0 6.0 3.0 1.0 1.0


Purification

www.zymoresearch.com


→ In step 7 the fragments were eluted in 12μL MQ and was stored on ice until use.
→ The concentration of GVP fragment with X/P sticky ends was 81.9 ng/μL, and was stored in fridge for restriction by MvaI/XhoI on Tuesday.


Ligation

A total amount of vector of 100ng was used in a 1:3 ratio with insert.

  • 3 uL Ligase buffer
  • 1 uL T4 Ligase
  • 3 uL GVP fragment 3300bp
  • 3 uL GVP fragment 2200bp
  • 4 uL plasmid J61035 XbaI/PstI
  • 8 uL GVP-insert (restricted with MvaI/XhoI)


Incubate:

  • 25°C 50min.
  • kept on ice for 10min.

Tranformation

  • add 10uL of the ligation product to 50uL competent E.coli TOP10 cells.

Incubate:

  • 30 min @ ice
  • 90 sec 42°C
  • 2 min @ ice
  • add 800uL LB-medium
  • incubate for 1 h at 37°C
  • plate on LB-amp100 plates


→ Negative control was MQ and positive was J61002-J23101.


Cultures

From the plates of last friday (growth from thursday to friday) two colonies of pMA-gvpL-insert, pMA-GlpF and pNL29, and one colony of pArsR-GVP in pSB2K3 were used to inoculate 6mL LB-amp100 and LB-kan50-IPTG medium for growth overnight.

→ The tubes were stored in a 37°C shaking waterbath.

Transporters

HmtA

Nieuwe poging om HmtA te krijgen. Via verschillende routes, PCR1 met F1 mut1rc en EcoRI cut pBAD met 742 f2 en PCR1 & 2.

Metal Accumulation

Vectors

Dry

April
MTWTFSS
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27 28 29 30
May
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11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June
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8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July
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6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
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3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
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7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
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5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
November
MTWTFSS
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2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30