Difference between revisions of "Part:BBa I13453"

(→‎TrpA Knockout Complementation by inducing arabinose promoter by British Columbia iGEM 2012)
 
(8 intermediate revisions by 4 users not shown)
Line 10: Line 10:
  
 
See this OpenWetWare article on [http://openwetware.org/wiki/Titratable_control_of_pBAD_and_lac_promoters_in_individual_E._coli_cells#pBAD_promotersOpenWetWare pBAD and lac promoters] for additional usage and biology information
 
See this OpenWetWare article on [http://openwetware.org/wiki/Titratable_control_of_pBAD_and_lac_promoters_in_individual_E._coli_cells#pBAD_promotersOpenWetWare pBAD and lac promoters] for additional usage and biology information
 +
 +
==2024 Squirrel-CHN supplementary applications==
 +
===Usage and Biology===
 +
First, we induced the expression of red fluorescent protein using the arabinose operon to verify its functionality in E. coli DH5α.
 +
Then, our team used the arabinose operon to induce the expression of the bacteriolytic proteins T4 holin and T4 lysozyme, with BBa_B0015 as the terminator. We recombined the fragments into the plasmid pET23b and transformed the constructed plasmid into E. coli DH5α.
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/5083/16.png" style="width: 500px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Fig 1.genetic circuit diagram</p >
 +
</div>
 +
</html>
 +
 +
===Potential application directions===
 +
Firstly, we performed sequence amplification of the arabinose operon, bacteriolytic protein T4 holin, and T4 lysozyme.
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/5083/6.png" style="width: 500px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Fig 2.Agarose gel electrophoresis to verify the sequence amplification</p >
 +
</div>
 +
</html>
 +
Secondly, we validated the functionality of the arabinose operon in E. coli DH5α. The results showed that as the concentration of arabinose increased, the Fluorescence/OD600 ratio increased, demonstrating that the arabinose operon can effectively induce the expression of the red fluorescent protein.
 +
 +
Finally, we introduced the suicide system into E. coli DH5α. The results indicated that under induction with 0.5 mM/L arabinose, the OD600 of the bacterial culture containing the arabinose operon suicide system was significantly lower than that of the control, proving the effectiveness of the suicide system.
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/5083/17.png" style="width: 500px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Fig 3.Verification of the Arabinose Operon Suicide System(A) Fluorescence/OD600 ratio of red fluorescent protein expression induced by different concentrations of arabinose operon(B) Growth of E. coli DH5α under 0.5 mM/L arabinose</p >
 +
</div>
 +
</html>
  
  
Line 51: Line 80:
 
<!-- -->
 
<!-- -->
  
−
===TrpA Knockout Complementation by inducing arabinose promoter by British Columbia iGEM 2012===
 
  
−
This part consists of an arabinose promoter, a strong RBS, and the TrpA coding gene. It is able to complement a metA knockout, and the growth rate appears to be proportional to the amount of arabinose that is added.
+
===MetA Knockout Complementation by inducing arabinose promoter by British Columbia iGEM 2012===
  
−
https://static.igem.org/mediawiki/parts/thumb/0/04/TrpA_Complementation_Test_No_fluor.png/800px-TrpA_Complementation_Test_No_fluor.png
 
  
−
To generate this graph we cultured an E. coli TrpA knockout transformed with this part in M9 minimal media with the indicated concentrations of arabinose, and measured the OD600 every 15 minutes. See <partinfo>BBa_K804009 for negative controls for this part as well as linked fluorescent data.
+
This part consists of an arabinose promoter, a strong RBS, and the MetA coding gene. It is able to complement a metA knockout, and the growth rate appears to be proportional to the amount of arabinose that is added.  
  
−
===MetA Knockout Complementation by inducing arabinose promoter by British Columbia iGEM 2012===
+
<p align=center> https://static.igem.org/mediawiki/parts/thumb/d/d3/MetA_complementation_no_fluor.png/800px-MetA_complementation_no_fluor.png</p>
  
−
This part consists of an arabinose promoter, a strong RBS, and the MetA coding gene. It is able to complement a metA knockout, and the growth rate appears to be proportional to the amount of arabinose that is added.
 
−
https://static.igem.org/mediawiki/parts/thumb/d/d3/MetA_complementation_no_fluor.png/800px-MetA_complementation_no_fluor.png
 
  
 
To generate this graph we cultured an E. coli MetA knockout transformed with this part in M9 minimal media with the indicated concentrations of arabinose, and measured the OD600 every 15 minutes.
 
To generate this graph we cultured an E. coli MetA knockout transformed with this part in M9 minimal media with the indicated concentrations of arabinose, and measured the OD600 every 15 minutes.
 +
 +
===TrpA Knockout Complementation by inducing arabinose promoter by British Columbia iGEM 2012===
 +
 +
This part consists of an arabinose promoter, a strong RBS, and the TrpA coding gene. It is able to complement a metA knockout, and the growth rate appears to be proportional to the amount of arabinose that is added.
 +
 +
https://static.igem.org/mediawiki/parts/thumb/0/04/TrpA_Complementation_Test_No_fluor.png/800px-TrpA_Complementation_Test_No_fluor.png
 +
 +
To generate this graph we cultured an E. coli TrpA knockout transformed with this part in M9 minimal media with the indicated concentrations of arabinose, and measured the OD600 every 15 minutes. See <partinfo>BBa_K804009 for negative controls for this part as well as linked fluorescent data.

Latest revision as of 06:31, 14 September 2024

Pbad promoter

PBad promoter from I0500 without AraC.


Usage and Biology

Has been used as a second promoter in a system containing BBa_I0500 (PBad+AraC). In this system, it showed behavior qualitatively indistinguishable from the BBa_I0500 copy of PBad. Has not been tested independent of AraC. A second part, BBa_I13458, should allow decoupling of PBad and AraC.

See this OpenWetWare article on [http://openwetware.org/wiki/Titratable_control_of_pBAD_and_lac_promoters_in_individual_E._coli_cells#pBAD_promotersOpenWetWare pBAD and lac promoters] for additional usage and biology information

2024 Squirrel-CHN supplementary applications

Usage and Biology

First, we induced the expression of red fluorescent protein using the arabinose operon to verify its functionality in E. coli DH5α. Then, our team used the arabinose operon to induce the expression of the bacteriolytic proteins T4 holin and T4 lysozyme, with BBa_B0015 as the terminator. We recombined the fragments into the plasmid pET23b and transformed the constructed plasmid into E. coli DH5α.

Fig 1.genetic circuit diagram

Potential application directions

Firstly, we performed sequence amplification of the arabinose operon, bacteriolytic protein T4 holin, and T4 lysozyme.

Fig 2.Agarose gel electrophoresis to verify the sequence amplification

Secondly, we validated the functionality of the arabinose operon in E. coli DH5α. The results showed that as the concentration of arabinose increased, the Fluorescence/OD600 ratio increased, demonstrating that the arabinose operon can effectively induce the expression of the red fluorescent protein.

Finally, we introduced the suicide system into E. coli DH5α. The results indicated that under induction with 0.5 mM/L arabinose, the OD600 of the bacterial culture containing the arabinose operon suicide system was significantly lower than that of the control, proving the effectiveness of the suicide system.

Fig 3.Verification of the Arabinose Operon Suicide System(A) Fluorescence/OD600 ratio of red fluorescent protein expression induced by different concentrations of arabinose operon(B) Growth of E. coli DH5α under 0.5 mM/L arabinose


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

For the 2009 iGEM competition, British_Columbia characterized BBa_I13453 in the context of a pBAD promoter family. For the results of this characterization, see here.

For the 2010 iGEM competition, Tec-Monterrey characterized BBa_I13453 again, using a construct of GFP reporter after the pBad promoter. The transfer function was modeled with a Hill equation.

Figure 1. Transfer function of BBa_I13453. Points represent individual measurements. The line is of a Hill equation fitted to our data.

(d[GFP]/dt)/OD600 = C+A*Xn/(Xn+Kn)

Experiment Characteristic Value
Transfer Function Basal rate (C) 1 d[GFP]/dt
Gain (A) 36 d[GFP]/dt
Hill coefficient (n) 2.16
Switch Point (K) 2.8 [ara] (µM)


MetA Knockout Complementation by inducing arabinose promoter by British Columbia iGEM 2012

This part consists of an arabinose promoter, a strong RBS, and the MetA coding gene. It is able to complement a metA knockout, and the growth rate appears to be proportional to the amount of arabinose that is added.

800px-MetA_complementation_no_fluor.png


To generate this graph we cultured an E. coli MetA knockout transformed with this part in M9 minimal media with the indicated concentrations of arabinose, and measured the OD600 every 15 minutes.

TrpA Knockout Complementation by inducing arabinose promoter by British Columbia iGEM 2012

This part consists of an arabinose promoter, a strong RBS, and the TrpA coding gene. It is able to complement a metA knockout, and the growth rate appears to be proportional to the amount of arabinose that is added.

800px-TrpA_Complementation_Test_No_fluor.png

To generate this graph we cultured an E. coli TrpA knockout transformed with this part in M9 minimal media with the indicated concentrations of arabinose, and measured the OD600 every 15 minutes. See BBa_K804009 for negative controls for this part as well as linked fluorescent data. Not understood