Difference between revisions of "BBa K731700 and BBa K731710 measurements"

(→‎PROTOCOL DEVELOPMENT:)
(→‎PROTOCOL DEVELOPMENT:)
Line 62: Line 62:
  
 
The same approach for the less bright protein (mCherry) is not exploitable since would have the effect of further reducing the emission peak intensity; the better way to have a lower influence on the emission peak due to the proximity to the excitation wavelenght is to use a wavelenght different from the emission peak for measurements (point with a bigger wasvelenght).
 
The same approach for the less bright protein (mCherry) is not exploitable since would have the effect of further reducing the emission peak intensity; the better way to have a lower influence on the emission peak due to the proximity to the excitation wavelenght is to use a wavelenght different from the emission peak for measurements (point with a bigger wasvelenght).
 +
 +
 +
<div style="text-align:center">
 +
 +
{|border = "1" cellpadding="5" cellspacing="0" align="centre"
 +
|
 +
|Excitation (nm)
 +
|Emission(nm)
 +
|-
 +
|mCherry
 +
|587
 +
|609
 +
|-
 +
|Venus
 +
|515
 +
|528
 +
|}
 +
</div>
 +
<p style="width:400px; margin-bottom:60px; text-align:justify "><em><strong>TABLE 1.</strong> '''Excitation and emission wavelengths from literature'''<br/>
 +
</em> </p>
 +
  
 
[[Image:Wls_K731710_520V.jpg]]  [[Image:Wls_K731700_570V.jpg]]
 
[[Image:Wls_K731710_520V.jpg]]  [[Image:Wls_K731700_570V.jpg]]

Revision as of 10:11, 22 September 2012

Hello world

In this page we are really proud to introduce you the protocol we (Giacomo and Anna) developed for the characterization transcriptional terminators effects on gene expression. (Tested on BBa_J64997)

The develop of this protocolcost us not only sleepless night but also sunny and beautiful weekend of trekking so take a hot cup of tea and read it all.


IN VIVO ANALYSIS

BEFORE STARTING:


  • In order to analyse terminators through this method is necessary to follow the entire procedure with both the platform without intervening terminator (control) and the platform with terminator (sample).


CELLS GROWTH AND SAMPLES PREPARATION (starting from a glycerol stock):


  • 100ul of cells (i.e. BL21(DE3) pLysS) tranformed with the platform (control or sample) in 10 ml fresh Luria Bertani medium (LB)

NOTE1:Antibiotic are at concentration of 0.1mg/ml ampicillin and 0.035mg/ml cloranphenicol

NOTE2:Four glycerol stocks for both control and sample will give you a good level of significance

  • Grow the cultures at 37°C in the termoshaker until optical density at 600nm (O.D.) ~ 0.4 (the O.D. at which you stop the sample is not important until it is major than 0.2.)
  • (chill on ice for the whole duration of this point) Dilute samples to OD 0.2 in order to have the same O.D. at the moment of induction.
    • x = 10ml*0.2 O.D./0.4 O.D. (where x are the ml of culture you need to take from the culture in the termoshaker)
    • LB = 10ml-x (where LB are the ml of LB you need to add to x to obtain OD 0.2)
  • Grow 37°C in the termoshaker until O.D. ~ 0.6.
  • Induce with 0.5mM Isopropyl β-D-1-thiogalactopyranoside (IPTG).
  • 3 hours induction at 37°C in the termoshaker.
  • Chill falcon on ice
  • Move 1ml of induced culture to an eppendorf.
  • Sonication (3 repetition of 10 s 50% amplitude sonication and 30 s of pause).
  • 1.5 min centrifuge (4000rpm).
  • Move supernatant from eppendorf to cuvette.
  • Add 1 mL buffer (i.e.: PBS)
  • Leave the cuvettes over night (O.N.) it the 4°C fridge
  • Fluorimetric measurements



PROTOCOL DEVELOPMENT:

Emission and Excitation parameter choise:

The emission and excitation parameters must be aimed at achievement of the maximum distance between the two points at which you take emission measurements and the maximum comparability in terms of fluorescence intensity according to the limits imposed by the two fluorescent proteins in use.


Example: The two proteins in use in BBa_K731700 and BBa_K731710 are mCherry and A206K Venus (mVenus). After excitation of the two proteins with standard parameters we note a huge difference in fluorescence intensity between the two proteins; moreover the proximity between the excitation wavelenght and the emission peak can easily influence the measurements. In this case a shift of the brighter protein (mVenus) excitation wavelenght toward smaller wavelenght can bring several benefits:

  • A decrease in fluorescene intensity
  • A larger distance between the excitation wavelenght and the emission peak


The same approach for the less bright protein (mCherry) is not exploitable since would have the effect of further reducing the emission peak intensity; the better way to have a lower influence on the emission peak due to the proximity to the excitation wavelenght is to use a wavelenght different from the emission peak for measurements (point with a bigger wasvelenght).


Excitation (nm) Emission(nm)
mCherry 587 609
Venus 515 528

TABLE 1. Excitation and emission wavelengths from literature


Wls K731710 520V.jpg Wls K731700 570V.jpg