Team:Bologna/Characterization

From 2009.igem.org

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= Plasmid copy number characterization =
= Plasmid copy number characterization =
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To test the ratio between the production of an 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 1429 promotor and the standard biobrick I13504.
+
To test the ratio between the production of an 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 1429 promotor and the standard biobrick I13504. From the Registry of Standard Biological Parts we knew that pSB1A2 is an high copy number plasmid while pSB3K3 is a low copy one, so the ideal ratio between their copy number is at least 10, but the best value that we reached with the spectrofluorimeter is about 3,3.
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PSB1A2 with high copy number plasmid and a low copy number were transformed in DH5alfa bacterial cells according to the standard protocol.  
PSB1A2 with high copy number plasmid and a low copy number were transformed in DH5alfa bacterial cells according to the standard protocol.  
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<br>
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One colony from each plate was picked up and let grow overnight in LB medium at 37°C. One milliliter for each of the two samples was collected by O/N cultures and spinned at 6000-8000 rpm for three minutes. The supernatant was harvested and the pellet resuspended. Slides were prepared for the fluorescence bacteria image acquisition.  
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One colony from each plate was picked up and let grow overnight in LB 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 fluorescence bacteria image acquisition.  
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Finally, images were elaborated with the fluorescence visualization software and these are the results:
Finally, images were elaborated with the fluorescence visualization software and these are the results:
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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 an high copy number plasmid (PSB1A2), the only different element was the promoter.
+
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 an high copy number plasmid (PSB1A2), the only different element was the promoter.  
-
<br><br>
+
 
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<br>
{|align="center"
{|align="center"
|[[Image:2547GFP open.png|center|450 px]]
|[[Image:2547GFP open.png|center|450 px]]
|[[Image:1429GFP_openloop_hc.png|center|450 px]]
|[[Image:1429GFP_openloop_hc.png|center|450 px]]
|}
|}
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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.
 +
The images, acquired during some repetitions of the test, was elaborated with the fluorescence visualization software (VIFluoR) giving out those results:
{|align="center"
{|align="center"
|[[Image:2500gfpy100cgn170esp0,25pomev6.png|center|thumbnail|385 px|Open loop GFP circuit with promoter J23100 (2547)]]
|[[Image:2500gfpy100cgn170esp0,25pomev6.png|center|thumbnail|385 px|Open loop GFP circuit with promoter J23100 (2547)]]
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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 1.8. Experimentally we have achieved the value of 1.2; for this reason we can say that this prove has gone well.
+
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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To be sure that the presence of the operator Ox doesn't significantly affect the GFP production we tested two open loop GFP circuits, one with the operator Ox and another without.
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With this trial we aimed to demonstrate that, in absence of LAC I protein, the operator Ox doesn't affect the bacterial production of GFP.
 +
<br>
 +
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 Ox. 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"
{|align="center"
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|[[Image:2547GFPOx_open.png|center|450 px]]
|[[Image:2547GFPOx_open.png|center|450 px]]
|}
|}
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Following the steps of the previous tests we obtained those results:
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Revision as of 18:34, 18 October 2009

ProvaBol2.png
HOME TEAM PROJECT SOFTWARE MODELING WET LAB PARTS HUMAN PRACTICE JUDGING CRITERIA


We decided to create and developed a biological circuit in which the TRANS-repressor, in absence of IPTG, can inhibit the synthesis of LacI repressor protein, warranting the production of GFP:

Circuit2.jpg


To do that we initially need to characterize some sub-circuits in order to obtain some information and values concerning the processes we were analyzing.

You can find here all our Wetlab Protocols

Plasmid copy number characterization

To test the ratio between the production of an 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 1429 promotor and the standard biobrick I13504. From the Registry of Standard Biological Parts we knew that pSB1A2 is an high copy number plasmid while pSB3K3 is a low copy one, so the ideal ratio between their copy number is at least 10, but the best value that we reached with the spectrofluorimeter is about 3,3.

1429GFP openloop hc.png
1429GFP openloop lc.png

PSB1A2 with 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 LB 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 fluorescence bacteria image acquisition.
Finally, images were elaborated with the fluorescence visualization software and these are the results:

High copy number plasmid (PSB1A2)
Low copy number plasmid (PSB3K3)



Promoter characterization

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 an high copy number plasmid (PSB1A2), the only different element was the promoter.


2547GFP open.png
1429GFP openloop hc.png

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:

Open loop GFP circuit with promoter J23100 (2547)
Open loop GFP circuit with promoter J23118 (1429)


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.

GFP production in absence/presence of operator Ox

With this trial we aimed to demonstrate that, in absence of LAC I protein, the operator Ox doesn't affect the bacterial production of GFP.
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 Ox. 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.

2547GFP open.png
2547GFPOx open.png

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