Difference between revisions of "Part:BBa K1412001"

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== '''What it is''' ==
 
== '''What it is''' ==
  
−
Composite part enables the chemotaxis of our engineering bacteria could be regulated by IPTG and L-Arabinose; pBAD and pLac promoters control the expression of Lac I and CheZ.
+
The expression of LacI and CheZ is under the control of pBAD and pLac promoter.
 +
This part enables us to reprogram chemotaxis of engineering ''E.coli CL-1'' (‘’CheZ’’ gene knocked out) thus we could regulate chemotaxis by IPTG and L-Arabinose.
  
 +
== '''What it does''' ==
  
 +
When L-Arabinose is added, pBAD promoter is activated, expressing repressor LacI, inhibiting the expression of CheZ. When IPTG is added, IPTG combines with LacI, forming a more stable complex, thus relief the inhibition of pLac and CheZ would be expressed to make bacteria regain the chemotaxis ability.
  
−
== '''What it does''' ==
 
  
−
when L-Arabinose is added, pBAD promoter is activated, expressing Lac I, inhibiting the expression of CheZ. When IPTG is added, IPTG combines with Lac I, forming a complex, thus relief the inhibition and the CheZ express, make the ability of chemotaxis recover.
+
[[File:Mechanism_of_BBa_K1412001.png‎|700px]]
  
 +
== '''How to use it''' ==
  
 +
Using IPTG as inducer and L-arabinose as inhibitor to govern the chemotaxis of the engineering bacteria.
  
−
== '''How to use it in their project''' ==
+
== '''Experimental data''' ==
  
−
Use IPTG as an inducer and L-Arabinose as an inhibitor to take control of the chemotaxis of the engineering bacteria.
+
===='''''More information in linking page of the following pictures.'''''====
 +
----
 +
'''Characterization of backbone effect'''<br>
 +
[[File:Curve_of_chemotaxis_diameter_under_different_antibiotics_concentrations.png |700px]]
  
 +
By adding chloramphenicol of gradient concentrations. iGEM14_[http://2014.igem.org/Team:XMU-China# XMU-China] found that the best chemotaxis ability will be got as the concentration of chloramphenicol is 50μg/ml.
  
−
== '''Experimental data''' ==
+
----
  
−
[[File:Curve_of_chemotaxis_diameter_over_time_under_different_IPTG_concentration_.png ]]
+
'''Characterization of IPTG effect'''<br>
 +
[[File:Curve_of_chemotaxis_diameter_over_time_under_different_IPTG_concentration_.png‎‎|700px ]]
  
−
[[File:Curve_of_chemotaxis_diameter_under_different_antibiotics_concentrations.png ]]
+
iGEM14_[http://2014.igem.org/Team:XMU-China# XMU-China] set the concentration of chloramphenicol at 50ug/ml. By adding gradient concentration of IPTG, they found that when the concentration of IPTG lies between 0.02 and 0.075, the chemotaxis distance is better than the others.
 +
[[File:Xmu_Project_Conic_Curve_IPTG_B.png |700px]]<br>
 +
As the concentration of IPTG keeps on decreasing as it spreads out. We spotted IPTG with its concentration a little higher than the optimum. We observed that reprogrammed bacteria swim to IPTG source which seemed that the bacteria were attracted by IPTG.
 +
----
 +
 
 +
'''Characterization of L-arabinose effect'''<br>
 +
[[File:Curve_of_chemotaxis_diameter_over_time_under_different_Ara_concentration_.jpg |700px]]
 +
 
 +
iGEM14_[http://2014.igem.org/Team:XMU-China# XMU-China] set the concentration of chloramphenicol at 50μg/ml and the concentration of IPTG at 0.025mM. They found that the most efficiency inhibition L-Arabinose concentration is 0.2% and chemotaxis ability will be a little stronger at the concentration of 0.02%.
 +
----
 +
 
 +
Using semisolid culture medium to draw geometry pattern<br>
 +
[[File:Characterization of part K1412001.JPG |700px]]
 +
 
 +
[[File:Characterization_of_forming_pattern_having_the_meaning_of_hyperbola.png |700px‎]]
 +
 
 +
 
 +
iGEM14_[http://2014.igem.org/Team:XMU-China# XMU-China] set the concentration of IPTG and L-Arabinose on the plate at 0.025mM and 0.02% respectively, then choose two points 0.5cm (up) [1cm (down)] away form each other on the plate. iGEM14_[http://2014.igem.org/Team:XMU-China# XMU-China] achieved their goal to command the ''CL-1'' to form one branch of hyperbolic pattern by spotting 0.25mM IPTG mixed with “CL-1” (√) and 1% L-Arabinose (X) on the semisolid culture medium.<br>
 +
 
 +
[[File:Xmu_Project_Conic_Curve_figure_mechainism.png |700px]]<br>
 +
Distances of the points on the critical line to the IPTG spot (focus), and the L-arabinose line (directrix) are in a fixed ratio (eccentricity). If the ratio is equal to 1, the critical line is a parabola. If the ratio is larger than 1, it is a branch of a hyperbola. Then we conducted experiment to verify the mechanism.<br>
 +
[[File:Xmu_Project_Conic_Curve_figure_hyperbola.png |700px]]<br>
 +
Left boundary of the colony is regarded as the critical line. We found that cells has the tendency to swim away from L-arabinose line which is an expected performance of the circuit.
 +
== '''protocol''' ==
 +
1.Transformation
 +
 
 +
2.Extract plasmids
 +
 
 +
3.Digestion
 +
 
 +
4.DNA gel electrophoresis
 +
 
 +
5.Gel Extraction
 +
 
 +
6.Ligation
 +
 
 +
7.Transformation
 +
 
 +
8.Extract plasmids
 +
 
 +
9.Digestion
 +
 
 +
10.DNA gel electrophoresis
 +
 
 +
11.Transform the plasmid into CL-1
  
−
[[File:Curve_of_chemotaxis_diameter_over_time_under_different_Ara_concentration_.png ]]
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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<!-- -->
 
<!-- -->
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'>
 +
 
 +
== '''Sequence and Features''' ==
 +
</span>
 
<partinfo>BBa_K1412001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1412001 SequenceAndFeatures</partinfo>
  
  
 +
 +
== '''Reference''' ==
 +
 +
[1] [http://www.ncbi.nlm.nih.gov/pubmed/21998392/ Sequential Establishment of Stripe Patterns in an Expanding Cell Population Chenli Liu et al.Science 334, 238 (2011);DOI: 10.1126/science.1209042]
 +
 +
[2][http://www.biomedsearch.com/nih/Reprogramming-bacteria-to-seek-destroy/20453864.html Reprogramming bacteria to seek and destroy an herbicide Joy Sinha1, Samuel J Reyes1 & Justin P Gallivan1*]
 +
 +
----
 +
 +
<I><B>More information, click here: [http://2014.igem.org/Team:XMU-China# XMU-China]
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K1412001 parameters</partinfo>
 
<partinfo>BBa_K1412001 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Latest revision as of 07:41, 22 October 2014

Endow the E.coli engineered strain(CL-1) the ability of chemotaxis


What it is

The expression of LacI and CheZ is under the control of pBAD and pLac promoter. This part enables us to reprogram chemotaxis of engineering E.coli CL-1 (‘’CheZ’’ gene knocked out) thus we could regulate chemotaxis by IPTG and L-Arabinose.

What it does

When L-Arabinose is added, pBAD promoter is activated, expressing repressor LacI, inhibiting the expression of CheZ. When IPTG is added, IPTG combines with LacI, forming a more stable complex, thus relief the inhibition of pLac and CheZ would be expressed to make bacteria regain the chemotaxis ability.


Mechanism of BBa K1412001.png

How to use it

Using IPTG as inducer and L-arabinose as inhibitor to govern the chemotaxis of the engineering bacteria.

Experimental data

More information in linking page of the following pictures.

Characterization of backbone effect
Curve of chemotaxis diameter under different antibiotics concentrations.png

By adding chloramphenicol of gradient concentrations. iGEM14_[http://2014.igem.org/Team:XMU-China# XMU-China] found that the best chemotaxis ability will be got as the concentration of chloramphenicol is 50μg/ml.

Characterization of IPTG effect
Curve of chemotaxis diameter over time under different IPTG concentration .png

iGEM14_[http://2014.igem.org/Team:XMU-China# XMU-China] set the concentration of chloramphenicol at 50ug/ml. By adding gradient concentration of IPTG, they found that when the concentration of IPTG lies between 0.02 and 0.075, the chemotaxis distance is better than the others. Xmu Project Conic Curve IPTG B.png
As the concentration of IPTG keeps on decreasing as it spreads out. We spotted IPTG with its concentration a little higher than the optimum. We observed that reprogrammed bacteria swim to IPTG source which seemed that the bacteria were attracted by IPTG.

Characterization of L-arabinose effect
Curve of chemotaxis diameter over time under different Ara concentration .jpg

iGEM14_[http://2014.igem.org/Team:XMU-China# XMU-China] set the concentration of chloramphenicol at 50μg/ml and the concentration of IPTG at 0.025mM. They found that the most efficiency inhibition L-Arabinose concentration is 0.2% and chemotaxis ability will be a little stronger at the concentration of 0.02%.

Using semisolid culture medium to draw geometry pattern

Error creating thumbnail: Invalid thumbnail parameters

700px‎


iGEM14_[http://2014.igem.org/Team:XMU-China# XMU-China] set the concentration of IPTG and L-Arabinose on the plate at 0.025mM and 0.02% respectively, then choose two points 0.5cm (up) [1cm (down)] away form each other on the plate. iGEM14_[http://2014.igem.org/Team:XMU-China# XMU-China] achieved their goal to command the CL-1 to form one branch of hyperbolic pattern by spotting 0.25mM IPTG mixed with “CL-1” (√) and 1% L-Arabinose (X) on the semisolid culture medium.

Xmu Project Conic Curve figure mechainism.png
Distances of the points on the critical line to the IPTG spot (focus), and the L-arabinose line (directrix) are in a fixed ratio (eccentricity). If the ratio is equal to 1, the critical line is a parabola. If the ratio is larger than 1, it is a branch of a hyperbola. Then we conducted experiment to verify the mechanism.
Xmu Project Conic Curve figure hyperbola.png
Left boundary of the colony is regarded as the critical line. We found that cells has the tendency to swim away from L-arabinose line which is an expected performance of the circuit.

protocol

1.Transformation

2.Extract plasmids

3.Digestion

4.DNA gel electrophoresis

5.Gel Extraction

6.Ligation

7.Transformation

8.Extract plasmids

9.Digestion

10.DNA gel electrophoresis

11.Transform the plasmid into CL-1


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]


Reference

[1] [http://www.ncbi.nlm.nih.gov/pubmed/21998392/ Sequential Establishment of Stripe Patterns in an Expanding Cell Population Chenli Liu et al.Science 334, 238 (2011);DOI: 10.1126/science.1209042]

[2][http://www.biomedsearch.com/nih/Reprogramming-bacteria-to-seek-destroy/20453864.html Reprogramming bacteria to seek and destroy an herbicide Joy Sinha1, Samuel J Reyes1 & Justin P Gallivan1*]

More information, click here: [http://2014.igem.org/Team:XMU-China# XMU-China]