Team:Bologna/Characterization

From 2009.igem.org

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(Promoter characterization)
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{{Template:BolognaTemplate}}
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We decided to create and developed a biological circuit in which the TRANS-repressor, in the absence of IPTG, can inhibit the synthesis of LacI repressor protein, warranting the production of GFP:
 
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[[Image:Circuit2OK.jpg|center|900px|]]
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=<font size="5" color="#000000"><b>Testing Circuit</b>=
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In order to test our T-REX device, we developed the following genetic circuit (Fig. 1):
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[[Image:circuit2OK.jpg|center|900px|thumb|<center>Figure 1 - Genetic Circuit to test CIS and TRANS' mRNA functionality</center>]]
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<font size="4">Before realizing the whole Testing Circuit, we decided to characterize its constitutive parts with intermediate circuits. </font>
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To do that we initially needed to characterize some sub-circuits in order to obtain some information and values concerning the processes we were analyzing.      
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=<font size="5" color="#000000"><b><i>pSB1A2</i> vs <i>pSB3K3</i></b>=
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You can find here all our [[Team:Bologna/WetlabProtocols|Wetlab Protocols]]
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* In order to identify the ratio between the high copy number the low to medium copy number plasmids, we analyzed the BBa_K201003 GFP production both on pSB1A2 and pSB3K3 (Fig. 4):
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''To test the ratio between the production of a high copy number plasmid (pSB1A2) and a low copy number one (pSB3K3), we assembled two circuits. The open loop GFP circuits are realized with a BBa_J23118 promoter and the standard biobrick I13504. From the Registry of Standard Biological Parts we knew that pSB1A2 is a high copy number plasmid while pSB3K3 is a low copy one, so the theoretical ratio between their copy number should be at least 10, but the highest value that we reached with the spectrofluorimeter was about 3,3.''
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= Plasmid copy number characterization =
 
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To test the ratio between the production of a high copy number plasmid (pSB1A2) and a low copy number one (pSB3K3), we assembled two circuits. The open loop GFP circuits are realized with a BBa_J23118 promoter and the standard biobrick I13504. From the Registry of Standard Biological Parts we knew that pSB1A2 is a high copy number plasmid while pSB3K3 is a low copy one, so the theoretical ratio between their copy number should be at least 10, but the highest value that we reached with the spectrofluorimeter was about 3,3.
 
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{|align="center"
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|[[Image:1429GFP_openloop_hc.png|center|450 px]]
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|[[Image:1429GFP_openloop_hc.png|center|450 px|thumb|<center><font face="Calibri" font size="4">Figure 4a - BBa_K201003 on pSB1A2</font></center>]]
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|[[Image:1429GFP_openloop_lc.png|center|450 px]]
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|[[Image:1429GFP_openloop_lc.png|center|450 px|thumb|<center><font face="Calibri" font size="4">Figure 4b - BBa_K201003 on pSB3K3</font></center>]]
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PSB1A2 with a high copy number plasmid and a low copy number were transformed in DH5alfa bacterial cells according to the standard protocol.  
PSB1A2 with a high copy number plasmid and a low copy number were transformed in DH5alfa bacterial cells according to the standard protocol.  
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|[[Image:1429i13504psb3k3y100cgn170esp1,4_v1.png|center|thumbnail|385 px|Low copy number plasmid (PSB3K3)]]
|[[Image:1429i13504psb3k3y100cgn170esp1,4_v1.png|center|thumbnail|385 px|Low copy number plasmid (PSB3K3)]]
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[[Image:boxplot_plas.png|center|thumbnail|700px|Box Plot of bacterium fluorescence. Max and minimum values are indicated by the horizontal bars.]]
[[Image:boxplot_plas.png|center|thumbnail|700px|Box Plot of bacterium fluorescence. Max and minimum values are indicated by the horizontal bars.]]
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[[Image:plasmidiGrafico2.png|center|thumbnail|700px]]
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=<font face="Calibri" font size="5" color="#000000"><b><i>BBa_J23100</i> vs <i>BBa_J23118</i></b>=
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<font face="Calibri" font size="4" color="#000000">
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= Promoter characterization =
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* In order to identify the ratio between BBa_J23100 and BBa_J23118 promoters, we analyzed the BBa_K079031 and BBa_K079032 GFP production on pSB1A2:
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In order to estimate the difference in strength of the two promoters J23100 (2547) and J23118 (1429), we realized two circuits. Both were composed by an open loop GFP mounted on a high copy number plasmid (pSB1A2), the only different element was the promoter.
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{|align="center"
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|[[Image:2547GFP_open_tag.png|center|450 px]]
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|[[Image:2547GFP_open_tag.png|center|450 px|thumb|<center><font face="Calibri" font size="4">Figure 5a - BBa_K079032 on pSB1A2</font></center>]]
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|[[Image:1429GFP_openloop_hc_tag.png|center|450 px]]
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|[[Image:1429GFP_openloop_hc_tag.png|center|450 px|thumb|<center><font face="Calibri" font size="4">Figure 5b - BBa_K079031 on pSB1A2</font></center>]]
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To do this we transformed those constructs in bacterial cells; we picked up a colony from each plate and we inoculated it in M9 medium. After growing all night at 37°C we took a milliliter of each sample and we measured their optical density; than we prepared slides for the fluorescence bacteria images acquisition, following the same steps of the previous test.  
To do this we transformed those constructs in bacterial cells; we picked up a colony from each plate and we inoculated it in M9 medium. After growing all night at 37°C we took a milliliter of each sample and we measured their optical density; than we prepared slides for the fluorescence bacteria images acquisition, following the same steps of the previous test.  
The images, acquired during some repetitions of the test, was elaborated with the fluorescence visualization software (VIFluoR) giving out those results:
The images, acquired during some repetitions of the test, was elaborated with the fluorescence visualization software (VIFluoR) giving out those results:
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From the registry of standard parts we learnt that the strengths of J23100 and J23118 are respectively 2547 and 1429, so the ratio between them is about 1.78. Experimentally we have achieved the value of 1.2; for this reason we can say that this prove has gone well.
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[[Image:boxplot.png|center|thumbnail|600px|Box Plot of bacterium fluorescence. Max and minimum values are indicated by the horizontal bars.]]
[[Image:boxplot.png|center|thumbnail|600px|Box Plot of bacterium fluorescence. Max and minimum values are indicated by the horizontal bars.]]
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[[Image:tabellaPromotoriGrafico2.png|center|thumbnail|600px]]
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<br>
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<font size="3">
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From the registry of standard parts we learnt that the strengths of J23100 and J23118 are respectively 2547 and 1429, so the ratio between them is about 1.78. Experimentally we have achieved the value of 1.2; for this reason we can say that this prove has gone well.
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=<font face="Calibri" font size="5" color="#000000"><b><i>Presence</i> vs <i>Absence</i> of LacI O2 natural operator</b>=
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* We needed to confirm that LacI natural operator O2 don't influence GFP production when LacI repressor is not present. We compare then the GFP expression level of BBa_K079032 and BBa_K201001
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= GFP production in absence/presence of operator O2 =
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With this trial we aimed to demonstrate that, in absence of LacI protein, the operator O2 doesn't affect the bacterial production of GFP.
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To do that we realized four circuits: the first was composed by the GFP protein under the control of the J23100 promoter mounted on the pSB1A2 plasmid; the second circuit differed from the first because it contained also the operator O2. Similarly the third and the fourth circuits differed only for the presence or the absence of the operator, but they mounted the J23118 promoter instead of the J23100.
 
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{|align="center"
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|[[Image:2547GFP_open_tag.png|center|450 px]]
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|[[Image:2547GFP_open_tag.png|center|450 px|thumb|<center><font face="Calibri" font size="4">Figure 6a - BBa_K079032 on pSB1A2</font></center>]]
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|[[Image:2547GFPO2_open_tag.png|center|450 px]]
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|[[Image:2547GFPO2_open_tag.png|center|450 px|thumb|<center><font face="Calibri" font size="4">Figure 6b - BBa_K201001 on pSB1A2</font></center>]]
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Following the steps of the previous tests we obtained those results:
Following the steps of the previous tests we obtained those results:
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Revision as of 01:49, 22 October 2009

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Contents

Testing Circuit



In order to test our T-REX device, we developed the following genetic circuit (Fig. 1): </html>

Figure 1 - Genetic Circuit to test CIS and TRANS' mRNA functionality

Before realizing the whole Testing Circuit, we decided to characterize its constitutive parts with intermediate circuits.

pSB1A2 vs pSB3K3



  • In order to identify the ratio between the high copy number the low to medium copy number plasmids, we analyzed the BBa_K201003 GFP production both on pSB1A2 and pSB3K3 (Fig. 4):

To test the ratio between the production of a high copy number plasmid (pSB1A2) and a low copy number one (pSB3K3), we assembled two circuits. The open loop GFP circuits are realized with a BBa_J23118 promoter and the standard biobrick I13504. From the Registry of Standard Biological Parts we knew that pSB1A2 is a high copy number plasmid while pSB3K3 is a low copy one, so the theoretical ratio between their copy number should be at least 10, but the highest value that we reached with the spectrofluorimeter was about 3,3.


Figure 4a - BBa_K201003 on pSB1A2
Figure 4b - BBa_K201003 on pSB3K3

PSB1A2 with a high copy number plasmid and a low copy number were transformed in DH5alfa bacterial cells according to the standard protocol.
One colony from each plate was picked up and let grow overnight in M9 medium at 37°C. One milliliter for each of the two samples was collected by O/N cultures and spinned at 8000 rpm for a minute; another milliliter was used for measuring the optical density and estimate the growth of the sample. The supernatant was harvested and the pellet resuspended. Slides were prepared for the acquisition of images of fluorescent bacteria.
Finally, images were elaborated with the fluorescence visualization software and these are the results:

High copy number plasmid (PSB1A2)
Low copy number plasmid (PSB3K3)
Box Plot of bacterium fluorescence. Max and minimum values are indicated by the horizontal bars.
PlasmidiGrafico2.png


BBa_J23100 vs BBa_J23118



  • In order to identify the ratio between BBa_J23100 and BBa_J23118 promoters, we analyzed the BBa_K079031 and BBa_K079032 GFP production on pSB1A2:


Figure 5a - BBa_K079032 on pSB1A2
Figure 5b - BBa_K079031 on pSB1A2



To do this we transformed those constructs in bacterial cells; we picked up a colony from each plate and we inoculated it in M9 medium. After growing all night at 37°C we took a milliliter of each sample and we measured their optical density; than we prepared slides for the fluorescence bacteria images acquisition, following the same steps of the previous test. The images, acquired during some repetitions of the test, was elaborated with the fluorescence visualization software (VIFluoR) giving out those results:
From the registry of standard parts we learnt that the strengths of J23100 and J23118 are respectively 2547 and 1429, so the ratio between them is about 1.78. Experimentally we have achieved the value of 1.2; for this reason we can say that this prove has gone well.

Open loop GFP circuit with promoter J23100 (2547)
Open loop GFP circuit with promoter J23118 (1429)
Box Plot of bacterium fluorescence. Max and minimum values are indicated by the horizontal bars.
TabellaPromotoriGrafico2.png



Presence vs Absence of LacI O2 natural operator



  • We needed to confirm that LacI natural operator O2 don't influence GFP production when LacI repressor is not present. We compare then the GFP expression level of BBa_K079032 and BBa_K201001


Figure 6a - BBa_K079032 on pSB1A2
Figure 6b - BBa_K201001 on pSB1A2


Following the steps of the previous tests we obtained those results:

Absence of O2
Presence of O2