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

Revision as of 08:02, 25 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 protocol cost us not only sleepless night but also wasted sunny and beautiful weekend so take a hot cup of tea and read it all.

IN VIVO ANALYSIS

BEFORE STARTING:

In order to analyze 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 seconds 50% amplitude sonication each 30 seconds).
1.5 min centrifuge (4000rpm).
Move supernatant from eppendorf to a cuvette.
Add 1 mL buffer (i.e.: phosphate buffer saline (PBS), 0.0067M PO4 without Ca or Mg, Cat number BE17-516F)
Leave the cuvettes over night (O.N.) it the 4°C refrigerator
Fluorimetric measurements

PROTOCOL DEVELOPMENT:

Emission and Excitation parameter choise:

The two proteins in use in BBa_K731700 and BBa_K731710 are mCherry and A206K Venus (mVenus), respectively upstream and downstream of the terminator. After excitation of the two proteins with standard parameters, we noticed a huge difference in fluorescence intensity between the two peaks, as mVenus emission peak was about 10 times stronger than the mCherry peak. Moreover, the two fluorescent proteins presented excitation and emission peaks very close to each other. This feature may cause unwanted interferences of the scattering effect caused by the excitation ray. // Due to the scattering effect caused by the closeness of the two peaks, interferences in the measure can occur.

In this case a shift of the brighter protein (mVenus) excitation wavelength toward smaller wavelength can bring two main benefits:

A decrease in emission peak's fluorescence intensity
A larger gap between the excitation wavelength and the emission peak's wavelength

Moreover, the less bright protein (mCherry) was measured at a slightly higher wavelength than the peak seen in the emission spectrum.

Here is a summary of the wavelengths used to take the measurements with BBa_K731700 and BBa_K731710:

	Standard Excitation (nm)	Standard Emission (nm)	Modified Excitation (nm)	Modified Emission (nm)
mCherry	587	609	587	615
mVenus	515	528	485	528

TABLE 1: Standard and modified excitation and emission wavelengths

CHART 1A/B: Emission scan of BBa_K731700 (A) and BBa_K731710 (B) with standard and modified excitation wavelenght. The measurements were taken with a Varian Cary Eclipse spectrofluorimeter, with 5nm slit and a voltage of 570 V (A) and 520 V (B))

Sonication of the samples:

In order to reduce the scattering due to excitation ray and thus to have a clearer signal, we decided to sonicate our sample before measure them. Both the improvements are due to the lower O.D. of a sonicated sample compared to a cell suspension sample. A higher O.D. causes a decrease in light intensity emitted by the fluorescent proteins that reach the sensor, a decrease in the exciting light that actually reach the fluorescent proteins and an increase in the excitation light that is reflected to the sensor (increase in scattering).

CHART 2: Comparison between a sonicated sample and a cells suspension sample

Test on different colonies:

The use of glycerol stock instead of O.N cultures makes the procedure easier and faster. However, glycerol stocks come from the same colony; to verify the consistency of our results we tested some different colonies from two different plates.

CHART 3: Fluorescence emission raw peaks of two cultures from colonies from different plates.
TABLE 2: The table shows the statistic analysis performed on the data in CHART 3. Neither the proteins peaks of the two plates nor their ratio are significantly different (the result of "0 min after sonication (A206K Venus plate 1 vs A206K Venus plate 2)" is considered a false positive since the other two times show no significant difference).

Different concentrations of inducing agent:

The use of different concentration of inducing agent should not influence the ratio between the fluorescence intensity of the two proteins. Nevertheless the inducing agent concentration may have influence on the quantity of proteins synthesized. We tested two different concentration of isopropyl β-D-1-thiogalactopyranoside (IPTG).

CHART 4: Raw emission peaks of coltures iduced with 0.1 mM or 0.5 mM IPTG. The measurements were taken just after induction (time 0) and after 90 minutes (time 1).
TABLE 3: The table shows the statistic analysis permormed on the data in CHART 4. There is no significant difference between the two samples with different IPTG concentration at time 0; significant differences are instead shown between the samples at induction time 90 min. The t-tests also show that the % increase in protein expression between 0.1mM e 0.5mM is the same in the two proteins.

O.D. equalization:

This point allows to have low standard deviation and variability in the data.

@@ Line 111: / Line 111: @@
 <p style="width:900px; margin-bottom:60px; text-align:justify"><em>
-<strong>CHART 3:</strong> ''Behavaior of different colonies.''
+<strong>CHART 3:</strong> ''Fluorescence emission raw peaks of two cultures from colonies from different plates.''<br/>
-<strong>TABLE 2:</strong> ''The table shows the statistic analysis performed on the data in CHART 3. Neither the proteins peaks of the two plates nor their ratio are significantly different (the result of ‘0 min after sonication (A206K Venus plate 1 vs A206K Venus plate 2)’ is considered a false positive since the other two times show no significant difference).''<br/>
+<strong>TABLE 2:</strong> ''The table shows the statistic analysis performed on the data in CHART 3. Neither the proteins peaks of the two plates nor their ratio are significantly different (the result of "0 min after sonication (A206K Venus plate 1 vs A206K Venus plate 2)" is considered a false positive since the other two times show no significant difference).''<br/>
 </em> </p>
 '''Different concentrations of inducing agent:'''
-The use of different concentration of inducing agent should not influence the ratio between the fluorescence intensity of the two proteins. Nevertheless we tested two different concentration to verify the veracity of this statement.
+The use of different concentration of inducing agent should not influence the ratio between the fluorescence intensity of the two proteins. Nevertheless the inducing agent concentration may have influence on the quantity of proteins synthesized. We tested two different concentration of isopropyl β-D-1-thiogalactopyranoside (IPTG).
-'''Example:'''Two identical cultures are grown in LB until O.D. 0.6 and then iduced with different IPTG concentration (0.1 mM and 0.5 mM). Starting from induction time 0, withdraw 1 mL of culture at predetermined times (in this case all the times after 90 min miss cause of overflow in mVenus fluorescence intensity). Every time you withdraw 1 mL of culture, you have to prepare the sample for the fluorimetric measurements according to the protol shown in the beginning of this page (start from the sonication point).
 [[Image:IPTGconcchart_inductiontime.jpg‎]][[Image:Statiptg.jpg]]
@@ Line 126: / Line 125: @@
 <p style="width:900px; margin-bottom:60px; text-align:justify"><em>
-<strong>CHART 4:</strong> ''Behavaior at different concentration of inducing agent (IPTG).''
+<strong>CHART 4:</strong> ''Raw emission peaks of coltures iduced with 0.1 mM or 0.5 mM IPTG. The measurements were taken just after induction (time 0) and after 90 minutes (time 1).''<br/>
-<strong>TABLE 3:</strong> ''The table shows the statistic analysis permormed on the data in CHART 4. There is no significant differences between the two samples at induction time 0 min; significant differences are instead shown between the samples at induction time 90 min. The t-tests also show that the % increase in protein expression between 0.1mM e 0.5mM is the same in the two proteins. ''<br/>
+<strong>TABLE 3:</strong> ''The table shows the statistic analysis permormed on the data in CHART 4. There is no significant difference between the two samples with different IPTG concentration at time 0; significant differences are instead shown between the samples at induction time 90 min. The t-tests also show that the % increase in protein expression between 0.1mM e 0.5mM is the same in the two proteins. ''<br/>
 </em> </p>