Difference between revisions of "Part:BBa K3552014"
 
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==Characterization==
 
==Characterization==
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Our new ptac promoter consists of 6 components, the reverse lac terminator, lacI, RBS, lac promoter and normal ptac promoter and lacO optimized. The new promoter comparing to the BBa_K180000 found in 2009 has a larger gradient of changing when comparing to the concentration of IPTG. In our experiment, we add different concentration of IPTG to constant promoter and received the value of florescence. We compared the value to the negative control and acquire the stable points to compare the data from the old ptac promoter.
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This year we improved the previous ptac promoter, part <partinfo>BBa_K180000</partinfo>, found in 2009 by reversing its lacI promoter, RBS, and terminator which will improve its yield of production. We also added three types of ribozymes, RiboJ, SccJ, and SarJ as an insulator to prevent the previous effect from the ptac promoter.  
  
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The main carbon source is the glycerol and the bacteria were cultured in M9 medium in order to simulate the expression of pilA. In this way, the result might be able to become a reference relating to our pilA production. The graph shows that after adding RiboJ, SccJ, and SarJ, the tangent of the line increased remarkably, referring to an ascent of the E.coli sensitivity of IPTG in low concentration. RiboJ has the best function on increasing the productivity of GFP among the five ribozymes.
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We first carried out an experiment in the culture medium with glucose as the source of carbon for the cultivation of bacteria in the M9 medium. In this experiment, we tested four ptac promoter, the old one, the new one, the one with RiboJ, and with SccJ. We convinced that all for ptac promoters are able to be activated when induced by IPTG, and as the concentration of IPTG increases, the expression of GFP genes promotes. Furthermore, the addition of two different types of ribozyme shows a prominent improvement of sensitivity towards the concentration of IPTG. At zero concentration of IPTG, all four promoter starts at a similar level,  as the concentration of IPTG increases, the gradient of the graph increased dramatically.
  
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We repeated the experiment in the culture medium with glucose as the source of carbon and gained similar results as the previous experiment. The data, in this manner, illustrate that the inducible promotor is robust in rising the yield in relatively low concentration. To our project, we have added RiboJ in front of our pilA genes to keep the gene in highly expression rate. In the following stage, we will apply these ribozymes on the generator transformation and better rise the production of pili generator. We hope this metamorphosis could generally increase the pili production.
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Afterward, according to our own medium, which utilizes glycerol as the only source of carbon, we attempted to simulate this situation for all five types of ptac promoter to investigate whether they can fit our experiments or not. Therefore, we substituted the medium to glycerol-M9 medium. The collected results imply that all five promoters can be activated obviously and the same as in glucose, promoters with ribozyme also obtain high sensitivity to the concentration of IPTG and dramatically increased.
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            [[File:T--LINKS_China--Figure 5 different ptac promoter.jpeg|600px]]
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These results confirmed the effect of our sensors under various types of carbon sources and maintain nearly no difference in practicability. We modified four new ptac promoter parts which are convinced to have higher sensitivity and adapt better to IPTG than the <partinfo>BBa_K180000</partinfo> discovered in 2009.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 13:47, 27 October 2020


ptac-new and lac repressor

Our new ptac promoter consists of 6 components, the reverse lac terminator, lacI, RBS, lac promoter and normal ptac promoter and lacO optimized. The new promoter comparing to the BBa_K180000 found in 2009 has a larger gradient of changing when comparing to the concentration of IPTG. In our experiment, we add different concentration of IPTG to constant promoter and received the value of florescence. We compared the value to the negative control and acquire the stable points to compare the data from the old ptac promoter.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

This year we improved the previous ptac promoter, part BBa_K180000, found in 2009 by reversing its lacI promoter, RBS, and terminator which will improve its yield of production. We also added three types of ribozymes, RiboJ, SccJ, and SarJ as an insulator to prevent the previous effect from the ptac promoter.

We first carried out an experiment in the culture medium with glucose as the source of carbon for the cultivation of bacteria in the M9 medium. In this experiment, we tested four ptac promoter, the old one, the new one, the one with RiboJ, and with SccJ. We convinced that all for ptac promoters are able to be activated when induced by IPTG, and as the concentration of IPTG increases, the expression of GFP genes promotes. Furthermore, the addition of two different types of ribozyme shows a prominent improvement of sensitivity towards the concentration of IPTG. At zero concentration of IPTG, all four promoter starts at a similar level, as the concentration of IPTG increases, the gradient of the graph increased dramatically.

Afterward, according to our own medium, which utilizes glycerol as the only source of carbon, we attempted to simulate this situation for all five types of ptac promoter to investigate whether they can fit our experiments or not. Therefore, we substituted the medium to glycerol-M9 medium. The collected results imply that all five promoters can be activated obviously and the same as in glucose, promoters with ribozyme also obtain high sensitivity to the concentration of IPTG and dramatically increased. 

           T--LINKS China--Figure 5 different ptac promoter.jpeg

These results confirmed the effect of our sensors under various types of carbon sources and maintain nearly no difference in practicability. We modified four new ptac promoter parts which are convinced to have higher sensitivity and adapt better to IPTG than the BBa_K180000 discovered in 2009.