Team:Groningen/Notebook/26 August 2009

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Latest revision as of 13:07, 26 August 2009

Wet

GVP Cluster

→ DONE Restriction of GVP and pSB1AC3-Lac/pBAD for assembly

→ DONE Gel purification of wanted fragments and nanodrop

→ DONE Clone GVP cluster behind Lac and Arabinose promoter for expression tests in pSB1AC3 plasmid

→ TODO Test promoter strenght compared to BBa_J23101 promoter (Sven)

→ TODO Enter sequences of constructs to Sandbox

Restriction for Assembly

The vector [http://partsregistry.org/wiki/index.php?title=Part:pSB1AC3 pSB1AC3] containing the LAC and pBAD inducible promoters were cut with PstI and SpeI to create correct ends for insert of GVP biobrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 BBa_I750016], which was cut with XbaI and PstI.

• 3 to 16μL plasmid in MQ (1.0μg)
• 13μL MQ (end volume of 20μL)
• 2μL Fast digest buffer
• 1μL PstI fast digest enzyme
• 1μL SpeI/XbaI fast digest enzyme

Restriction was kept at 37C for 30 min. and put on ice until used for gel purification.

Purification

→ From left to right: 1kB ladder, pSB1AC3-LAC ,Empty Slot, pSB1AC3-pBAD-AraC , Empty Slot, GVP

→ The band for the pSB1AC3-LAC vector was expected to be at ~3700bp (red line), but instead it was at ~2500bp.

→ Comparison with the gel of Frans (constructed pSB1AC3-LAC) explained the band at a lower position (also seen on his gel), but not why it is so small. One option is use of pSB3K3 plasmid instead of pSB1AC3 plasmid, this would still give a band of ~3300bp and not ~2500bp!

A "Agarose Gel DNA Extraction Kit" [http://www.roche-applied-science.com/pack-insert/1696505a.pdf Standard Protocol] from Roche Applied Science.

→ In step ten, 30μL MQ was added to the dry pellet and incubated at room temperature.

Concentrations

Ligation

A total amount of vector of 50ng was used (GVP) in a 1:2 ratio with insert.

(1:2) (Done twice, old and new cut GVP insert)

• 4 uL Ligase buffer
• 2 ul T4 Ligase
• 8 uL plasmid pSB1AC3-LAC/pBAD digested with PstI and SpeI
• 8 uL insert GVP restricted with XbaI and PstI

(1:2)

• 4 uL Ligase buffer
• 2 ul T4 Ligase
• 8 uL plasmid pSB3K3-L digested with PstI and SpeI
• 8 uL insert GVP restricted with XbaI and PstI

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/LB-kan₅₀ plates

→ Positive control was BBa_J61002-J23101 plasmid, and negative control was MQ.

Transporters

Metal Accumulation

MBP-ArsR

• Transform ligation with inducible promotors
• Prepare glycerol stock without promotors

Vectors

Dry