Team:Groningen/Notebook/10 August 2009

GVP Cluster
Restriction for Assembly

The vector pSB1AC3 containing the high, medium and low constitutive promoters were cut with PstI and SpeI to create correct ends for insert of GVP biobrick 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.

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&deg;C for 5min
 * Touchdown 10x
 * denat 95&deg;C for 30sec
 * anneal 62-50&deg;C for 30sec
 * elong 72&deg;C for 30sec
 * Cycles 20x
 * denat 95&deg;C for 30sec
 * anneal 58&deg;C for 30sec
 * elong 72&deg;C for 30sec
 * Final elong 72&deg;C for 5min
 * Hold 4&deg;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&deg;C for 5min
 * Touchdown 10x
 * denat 98&deg;C for 30sec
 * anneal 65-58&deg;C for 1min:30sec
 * elong 72&deg;C for 1min:20sec
 * Cycles 25x
 * denat 98&deg;C for 30sec
 * anneal 59&deg;C for 1min:30sec
 * elong 72&deg;C for 30sec
 * Final elong 72&deg;C for 5min
 * Hold 4&deg;C infinite


 * Run on 1%Agarose gel.

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.
 * 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.)
 * 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).
 * ✅ The uptake formulas on the transport page should be clearer (and less fidling with units).
 * ✅ Vmax should have a lower-case v to distinguish rates and volumes.
 * ✅(?) Infobox z-order not correct in IE (next to a graph as on the transport page at least).
 * ✅ Missing info section should be updated on the transport page.
 * ✅ ArsD can be eliminated from the text a bit more.
 * ✅ Variables should be clarified (especially) on the accumulation page.
 * ✅ MT reactions should be clarified/removed.
 * Tip of gas vesicle in the diagram on the gas vesicle page should be clearly shown as a magnification.
 * ✅ Formulas on the gas vesicle page should be removed from the text and put in equation boxes (or something).
 * ✅ "... the density [of (wild-type) E. coli] stays within"
 * ✅ The range of densities 1100 +- 3% (instead of natural language description).