Difference between revisions of "Part:BBa K864009:Experience"
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'''iGEM Team Uppsala University 2012'''
 
'''iGEM Team Uppsala University 2012'''
  
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The copy number of a cousin of pSB4A15Iq, the <partinfo>BBa_K864001</partinfo>, has been estimated by three methods all pointing to it being maintained at a consistent low copy number in E coli cells. These parts are identical, except for the presence of a lacIq cassette, and it is therefore strongly suggested they will behave similar. Read about [[Part:BBa_K864001:Experience|pSB4C15]] for details.  
+
The copy number of a cousin of pSB4C15Iq, the <partinfo>BBa_K864001</partinfo>, has been estimated by three methods all pointing to it being maintained at a consistent low copy number in E coli cells. These parts are identical, except for the presence of a lacIq cassette, and it is therefore strongly suggested they will behave similar. Read about [[Part:BBa_K864001:Experience|pSB4C15]] for details.  
  
 
*Measurment of fluorescence by FACS flow cytometry
 
*Measurment of fluorescence by FACS flow cytometry
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To investigate the functionality of lacIq repression and the effect of IPTG induction in the pSB4x15Iq backbones, three common lac-repressed promoters were assembled with red fluorescent protein (RFP) in the pSB4C15 backbone and grown with and without IPTG. Flourescence was measured  with a fluorescence activated cell sorter (FACS).
 
To investigate the functionality of lacIq repression and the effect of IPTG induction in the pSB4x15Iq backbones, three common lac-repressed promoters were assembled with red fluorescent protein (RFP) in the pSB4C15 backbone and grown with and without IPTG. Flourescence was measured  with a fluorescence activated cell sorter (FACS).
  
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Cassettes of the PlacI, PLlacO (<partinfo>R0011</partinfo>) and PT5lac (<partinfo>K592008</partinfo>) promoters and the pUCori-lacI and RFP were assembled in the pSB4C15Iq backbone, and transformed into E coli DH5alpha. Single clones of these were inoculated in 2 mL LB media with chloramphenicol (12 µg/mL) and with or without IPTG (0.5 mM). Cultures were grown shaking at 37° C for 18 hours, and were visibly confirmed to have grown at steady state for at least 4 hours.  
+
Cassettes of the PlacI, PLlacO (<partinfo>R0011</partinfo>) and PT5lac (<partinfo>K592008</partinfo>) promoters and the pUCori-lacI were assembled with RFP in the pSB4C15Iq backbone, and transformed into E coli DH5alpha. Single clones of these were inoculated in 2 mL LB media with chloramphenicol (12 µg/mL) and with or without IPTG (0.5 mM). Cultures were grown shaking at 37° C for 18 hours, and were visibly confirmed to have grown at steady state for at least 4 hours.  
  
 
[[Image:Lacirepression.png|300px|thumb|Relative fluorescence of RFP with different promters in pSB4C15Iq, with and without IPTG induction (0.5 mM). Fluorescence is in arbitrary units. ]]
 
[[Image:Lacirepression.png|300px|thumb|Relative fluorescence of RFP with different promters in pSB4C15Iq, with and without IPTG induction (0.5 mM). Fluorescence is in arbitrary units. ]]
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!  
 
!  
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!cellspan=2|Fluorescence (arbitrary units)
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!Fluorescence  
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'''Conclusions'''
 
'''Conclusions'''
  
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Results of fluorescence measurments, plasmid yield and color development on plates all point to pSB4C15 being a true low copy backbone. It is strongly suggested that this result is also valid for the pSB4x15Iq series, since they only differ in their resistance cassette. The IPTG repression data demonstrates that the pSB4C5Iq backbone is capable of tightly repressing promoters on the same plasmid, whether it is present in high or low copy number. It also demonstrates that expression can be induced to significant levels by addition of IPTG.   
+
Results of fluorescence measurments, plasmid yield and color development on plates all point to pSB4C15 being a true low copy backbone. It is strongly suggested that this result is also valid for the pSB4x15Iq series, since they share an identical origin. The IPTG repression data demonstrates that the pSB4C5Iq backbone is capable of tightly repressing promoters on the same plasmid, whether it is present in high or low copy number. It also demonstrates that expression can be induced to significant levels by addition of IPTG.   
  
 
|};
 
|};

Revision as of 13:52, 27 September 2012

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K864009

User Reviews

UNIQ21d8582a0316da37-partinfo-00000000-QINU

No review score entered. User:agynna

iGEM Team Uppsala University 2012

The copy number of a cousin of pSB4C15Iq, the BBa_K864001, has been estimated by three methods all pointing to it being maintained at a consistent low copy number in E coli cells. These parts are identical, except for the presence of a lacIq cassette, and it is therefore strongly suggested they will behave similar. Read about pSB4C15 for details.

  • Measurment of fluorescence by FACS flow cytometry
  • Measurment of plasmid yields from liquid cultures
  • Visual color development of colonies on plates

The functionality of the lacIq repression, and possibility to induce transcription by IPTG has been assessed for three common lacI-repressed promoters in pSB4C15Iq. The results demonstrates tight repression with a possibility of induction.

Copy number measurement by flow cytometry

Relative fluorescence of RFP with different promters in pSB4C5Iq, with and without IPTG induction (0.5 mM). Fluorescence is in arbitrary units.

E coli MG1655 was transformed with plasmids of different backbones with the J04450 standard RFP cassette. Liquid cultures were analysed by fluorescence with a Fluorescence activated cell sorter (FACS), quantitivly measuring the fluorescence of individual cells. Due to the active native lacI repression system in the bacteria, the experiment was performed with and without IPTG for promoter induction.

Read about pSB4C15 for other measurements.

LacI repression

To investigate the functionality of lacIq repression and the effect of IPTG induction in the pSB4x15Iq backbones, three common lac-repressed promoters were assembled with red fluorescent protein (RFP) in the pSB4C15 backbone and grown with and without IPTG. Flourescence was measured with a fluorescence activated cell sorter (FACS).

Cassettes of the PlacI, PLlacO (BBa_R0011) and PT5lac (BBa_K592008) promoters and the pUCori-lacI were assembled with RFP in the pSB4C15Iq backbone, and transformed into E coli DH5alpha. Single clones of these were inoculated in 2 mL LB media with chloramphenicol (12 µg/mL) and with or without IPTG (0.5 mM). Cultures were grown shaking at 37° C for 18 hours, and were visibly confirmed to have grown at steady state for at least 4 hours.

Relative fluorescence of RFP with different promters in pSB4C15Iq, with and without IPTG induction (0.5 mM). Fluorescence is in arbitrary units.

Samples were equilibrated in PBS solution at 1:160 dilution for 1 hour, and then measured in a BD Biosciences FACSaria II . 10^5 cells of each sample were individually measured and averaged, with dead and other non-flourescent cells excluded. Since the purpose of the experiment was to investigate overall repression functionality and the possibility of induction, the samples of with PlacI, PT5lac and PLlacO can be considered a triplicate measurement.

Fluorescence (arbitrary units)
-IPTG +IPTG
pUC-PlacI 0.012 0.97
PLlacO 0.17 4.26
PT5lac 0.06 3.58
PlacI 0.07 1.52

Conclusions

Results of fluorescence measurments, plasmid yield and color development on plates all point to pSB4C15 being a true low copy backbone. It is strongly suggested that this result is also valid for the pSB4x15Iq series, since they share an identical origin. The IPTG repression data demonstrates that the pSB4C5Iq backbone is capable of tightly repressing promoters on the same plasmid, whether it is present in high or low copy number. It also demonstrates that expression can be induced to significant levels by addition of IPTG.

;

UNIQ21d8582a0316da37-partinfo-00000005-QINU