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
• 90 sec 42°C
• 2 min @ ice
• add 800uL LB-medium
• incubate for 1 h at 37°C
• plate on LB-amp₅₀/chlo₂₀ 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 (-80 no.12)

→ E.coli TOP10 med. const. promoter (-80 no.11)

→ E.coli TOP10 low const. promoter (-80 no.10)

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. Also the loading dye is problematic because it runs @ ~150-200bp. Therefore next time use Orange Dye.

• 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

Jasper talked with Wilfred about his comments on the modelling work and worked on integrating (indirect) regulation of ArsB by arsenic with our model (which is still a work in progress). Some of Wilfred's comments:

• We can still simplify some formulas by reusing common parts. For the moment we won't do this because it's still in flux and could change at any time.
• TODO It would be nice if we could look at how long it would take for our gas vesicles to get filled with gas. (And take the concentration of gas in the medium into account.)
• TODO We should clarify what "resting" means. And in fact we'd love to know ourselves, so we're going to ask this tomorrow (but we've assumed that it means that they're not supposed to divide).
• DONE The uptake formulas on the transport page should be clearer (and less fidling with units).
• DONE V_max should have a lower-case v to distinguish rates and volumes.
• DONE(?) Infobox z-order not correct in IE (next to a graph as on the transport page at least).
• DONE Missing info section should be updated on the transport page.
• DONE ArsD can be eliminated from the text a bit more.
• DONE Variables should be clarified (especially) on the accumulation page.
• DONE MT reactions should be clarified/removed.
• TODO Tip of gas vesicle in the diagram on the gas vesicle page should be clearly shown as a magnification.
• DONE Formulas on the gas vesicle page should be removed from the text and put in equation boxes (or something).
• DONE "... the density [of (wild-type) E. coli] stays within"
• DONE The range of densities 1100 +- 3% (instead of natural language description).