Team:Groningen/Notebook/10 August 2009

From 2009.igem.org

Wet

GVP Cluster

Restriction for Assembly

The vector [http://partsregistry.org/wiki/index.php?title=Part:pSB1AC3 pSB1AC3] containing the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 high], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 medium] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 low] constitutive 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 10μL plasmid in MQ (1.0μg)
• 6 to 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

Concentrations

Ligation

(1:3)

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

(1:6)

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

Incubate:

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

Tranformation

• add 10uL of the pSB1AC3-GVP ligation product to 50uL competent E.coli TOP10 cells.

Incubate:

• 30 min @ ice
• 50 sec 42°C
• 2 min @ ice
• add 800uL LB-medium
• incubate for 1 h at 37°C
• plate on LB-amp₅₀ plates

Over Night Cultures

Over night cultures in 4mL LB-amp₁₀₀ medium were prepared from the following glycerol stock:

→ E.coli TOP10 high const. promoter

→ E.coli TOP10 med. const. promoter

→ E.coli TOP10 low const. promoter

and put in the 37°C waterbath at 200 rpm.

Transporters

Metal Accumulation

• Check colonies of E. coli + pGB68 (mymT)
  • There were colonies on the MymT plates (a lot on the + control, but also 2 on - control...), 4 colonies were picked for o/n culture and colony PCR.
  • Colony PCR as done for pSB1AC3-HML+RFP and pSB3K3-HML+RFP though the PCR program was slightly modified:

Initial denat 95°C for 5min

Touchdown 10x

denat 95°C for 30sec

anneal 62-50°C for 30sec

elong 72°C for 30sec

Cycles 20x

denat 95°C for 30sec

anneal 58°C for 30sec

elong 72°C for 30sec

Final elong 72°C for 5min

Hold 4°C infinite

• Run on 1%Agarose gel.

• • Contents: Marker-MymT(colony)1-MymT2-MymT3-MymT4
  • On the gel a very faint band may be seen at the lanes with MymT3 and MymT4.. But there are too faint to be sure about there nature, it may also be possible that there unspecific products

• Amplify SmtA and SmtA-GST by PCR
  • Use protocol as used for amplification of SmtA and SmtA-GST written on 7 August 2009.
  • PCR program was modified to get the following:

Initial denat 95°C for 5min

Touchdown 10x

denat 98°C for 30sec

anneal 65-58°C for 1min:30sec

elong 72°C for 1min:20sec

Cycles 25x

denat 98°C for 30sec

anneal 59°C for 1min:30sec

elong 72°C for 30sec

Final elong 72°C for 5min

Hold 4°C infinite

• Run on 1%Agarose gel.

Vectors

Dry