Difference between revisions of "Part:BBa K374006:Experience"

(→‎Positive feedback antitermination on double reporter plasmid)
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
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how you used this part and how it worked out.
 
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=Characterization of BBa_K374006=
 
=Characterization of BBa_K374006=
  
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* If it was possible to trigger a positive feedback mechanism of N protein, by varying the promoter strength with the synthetic promoter library (SPL) and thus antitermination and constant expression of the down stream RFP.  
 
* If it was possible to trigger a positive feedback mechanism of N protein, by varying the promoter strength with the synthetic promoter library (SPL) and thus antitermination and constant expression of the down stream RFP.  
 
* If it was possible to trigger this positive feed back mechanism by inducing the N protein from a second plasmid insert with the pBAD promoter and the N protein.
 
* If it was possible to trigger this positive feed back mechanism by inducing the N protein from a second plasmid insert with the pBAD promoter and the N protein.
 +
 +
The following test strains were constructed, see below. Of the original colonies selected from the transformation of Serie A constructs. 11 were selected for transformation with pAT01, the inducible pBAD+Nprotein plasmid. This series of 11 strains were succesfull transformed with pAT01, serie N.  All the strains were tested both with florescence microscope, flourometer and a microfermentor system.
  
 
=== Results ===
 
=== Results ===
 +
The following experiments were preformed to verify the functionality of the biobrick.
  
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'''Florescence microscope'''
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The results for
 +
[[Image:transformations.PNG|center|thumb|400px|blblbl]]
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 +
 +
We did a series of restreaks of the successful serie A transformations described above. These were used for Biolector experiments, following we looked at the colonies in the microscope to verify BioLector data and construct stability.
 +
 +
The efficiency rate were drastically decreased, as was the consistency regarding the terminator efficiency. Most of the colonies that had undetectable levels of GFP have gained a mutation that prevents expression of a functional GFP, as they had detectable levels when isolated, expect two colonies in each construct that was selected for low GFP expression.  Only one colony (B10) of the selected and restreaked colonies from the B and C constructs expressed trigger mechanism and expressed RFP, see the table below.
 +
 +
[[Image:restreaks.PNG|center|thumb|400px|bjdso]]
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'''Micro fermentor'''
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Overnigth cultures of both series A and N were run in the Micro fermentor and serie N were induced with arabinose after approx. 4 hours to induce N protein expression, to verify if the trigger mechanism seen from the florescence microscope (see below) could be induced.  Of the 9 cultures only one of the E-constructs gave a GFP signal (strong promoter – weak Terminator – no N protein), but non of the constructs expressed RFP. Data not shown.
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 +
'''Fluorometer'''
 +
At the same time the overnight cultures were diluted and new overnight cultures were made with a reference and also induced with arabinose to test if a difference in the RFP expression could be seen.  Non of the cultures expressed RFP. Data not shown.
  
  
 
==== Conclusionc and Recommendations ====
 
==== Conclusionc and Recommendations ====
  
 +
From the initial verification by florescence microscopy, our constructs seems to work and have the expected functionality. 10 colonies from each construct, except C that did only have one colony, were selected for further verification in the BioLeactor.
 +
 +
It was not possible to verify the trigger or inducible mechanism
 +
 +
It was only possible to detect both GFP and RFP when examining the plated coloniestransformations with
  
 +
Summary on the different experiments:
 +
It have been shown in literature that the nut-site terminator position can be synthetically designed an function in vitro, it has not before been tested with as short distance between the two functional parts as we have in our constructs.
 +
The constructs
 +
We have successfully constructed the testplasmids and verified the length by gels and have shown that the cells can express variying levels of GFP and RFP in relation to the SPL-promoter strategy. could expressWe have results that indicate that
 +
Antiterminator interaction nut-site N protein
 +
Toxicity of high N expression
 +
GFP toxicity?
  
  

Revision as of 23:47, 27 October 2010

Characterization of BBa_K374006

Positive feedback antitermination on double reporter plasmid

The iGEM DTU team 2010 have submitted this part, after using it in the development of a genetic bistable switch.

Aim and strategy

The aim of the characterization was to asses if this part together with BBa_K374005 could have an antiterminating function that could be used as a regulatory mechanism. We characterized the functionality of the part in the following construct.
The test plasmid was constructed to biobrick standards, for more information see our wiki, GFP and RFP was used as reporter proteins. The distance from the boxB site in the nut site (marked with a grey triangle) to the terminator was 26bp. The RBS sites was the BB standard RBS BBa_B0034, except for the N protein where the natural RBS site was used. 5 constructs where produced named from A to E, with three different terminator strengths and references with out terminator and N protein.

we tested if:

  • If it was possible to trigger a positive feedback mechanism of N protein, by varying the promoter strength with the synthetic promoter library (SPL) and thus antitermination and constant expression of the down stream RFP.
  • If it was possible to trigger this positive feed back mechanism by inducing the N protein from a second plasmid insert with the pBAD promoter and the N protein.

The following test strains were constructed, see below. Of the original colonies selected from the transformation of Serie A constructs. 11 were selected for transformation with pAT01, the inducible pBAD+Nprotein plasmid. This series of 11 strains were succesfull transformed with pAT01, serie N. All the strains were tested both with florescence microscope, flourometer and a microfermentor system.

Results

The following experiments were preformed to verify the functionality of the biobrick.

Florescence microscope The results for

blblbl


We did a series of restreaks of the successful serie A transformations described above. These were used for Biolector experiments, following we looked at the colonies in the microscope to verify BioLector data and construct stability.

The efficiency rate were drastically decreased, as was the consistency regarding the terminator efficiency. Most of the colonies that had undetectable levels of GFP have gained a mutation that prevents expression of a functional GFP, as they had detectable levels when isolated, expect two colonies in each construct that was selected for low GFP expression. Only one colony (B10) of the selected and restreaked colonies from the B and C constructs expressed trigger mechanism and expressed RFP, see the table below.

bjdso

Micro fermentor Overnigth cultures of both series A and N were run in the Micro fermentor and serie N were induced with arabinose after approx. 4 hours to induce N protein expression, to verify if the trigger mechanism seen from the florescence microscope (see below) could be induced. Of the 9 cultures only one of the E-constructs gave a GFP signal (strong promoter – weak Terminator – no N protein), but non of the constructs expressed RFP. Data not shown.

Fluorometer At the same time the overnight cultures were diluted and new overnight cultures were made with a reference and also induced with arabinose to test if a difference in the RFP expression could be seen. Non of the cultures expressed RFP. Data not shown.


Conclusionc and Recommendations

From the initial verification by florescence microscopy, our constructs seems to work and have the expected functionality. 10 colonies from each construct, except C that did only have one colony, were selected for further verification in the BioLeactor.

It was not possible to verify the trigger or inducible mechanism

It was only possible to detect both GFP and RFP when examining the plated coloniestransformations with

Summary on the different experiments: It have been shown in literature that the nut-site terminator position can be synthetically designed an function in vitro, it has not before been tested with as short distance between the two functional parts as we have in our constructs. The constructs We have successfully constructed the testplasmids and verified the length by gels and have shown that the cells can express variying levels of GFP and RFP in relation to the SPL-promoter strategy. could expressWe have results that indicate that Antiterminator interaction nut-site N protein Toxicity of high N expression GFP toxicity?


User Reviews

UNIQ4be271db7136eb3e-partinfo-00000001-QINU

BBa_K374006 1 Not understood iGEM DTU 2010

We have characterized this part.

;


UNIQ4be271db7136eb3e-partinfo-00000003-QINU