Difference between revisions of "Part:BBa K1412001"

Revision as of 08:42, 12 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 promoters. 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.

How to use it

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

Experimental data

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*]

@@ Line 5: / Line 5: @@
 == '''What it is''' ==
-This part enables the chemotaxis of our engineering ''E.coli CL-1''(CheZ knocked out) to be reprogrammed which could be regulated by IPTG and L-Arabinose. The expression of LacI and CheZ is under the control of pBAD and pLac promoters.
+The expression of LacI and CheZ is under the control of pBAD and pLac promoters.
+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 complex, thus relief the inhibition and the CheZ express, make the ability of chemotaxis recover.
+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.
@@ Line 17: / Line 17: @@
 == '''How to use it''' ==
-Use IPTG as an inducer and L-Arabinose as an inhibitor to take control of the chemotaxis of the engineering bacteria.
+Using IPTG as inducer and L-arabinose as inhibitor to govern the chemotaxis of the engineering bacteria.
 == '''Experimental data''' ==
@@ Line 24: / Line 23: @@
 ===='''More information in linking page of the following pictures.'''====
+Characterization of backbone effect
 [[File:Curve_of_chemotaxis_diameter_under_different_antibiotics_concentrations.png |700px]]
+By adding chloramphenicol of gradient concentrations. iGEM14_XMU-China found that the best chemotaxis ability will be got as the concentration of chloramphenicol is 50μg/ml.
-By adding chloramphenicol of different concentrations. Team XMU2014 found the best chemotaxis as the concentration is 50μg/ml
 ----
+Characterization of IPTG effect
 [[File:Curve_of_chemotaxis_diameter_over_time_under_different_IPTG_concentration_.png‎‎|700px ]]
+iGEM14_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.
-Team XMU2014 set the concentration of chloramphenicol at 50ug/ml. By adding IPTG of different concentration levels, Team XMU2014 found that when the concentration of IPTG lies between 0.02 and 0.075, the chemotaxis diameter is better than the others.
 ----
+Characterization of L-arabinose effect
 [[File:Curve_of_chemotaxis_diameter_over_time_under_different_Ara_concentration_.jpg|700px]]
+iGEM14_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%.
-iGEM14_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 concentration of L-Arabinose is 0.2% and best inducing concentration is 0.02%.
 ----
+Using semisolid culture medium to draw geometry pattern
 [[File:Characterization of part K1412001.JPG |700px]]
 [[File:Characterization_of_forming_pattern_having_the_meaning_of_hyperbola.png |700px‎]]
-iGEM14_XMU-China set the concentration of IPTG and L-Arabinose on the plate at 0.025mM and 0.02% deliberately, then choose two points 0.5cm (up) [1cm (down)] away form each other on the plate. Team XMU2014 achieved the goal of command the ''CL-1'' to form hyperbolic curve by doting 0.25mM IPTG（√） and 1% L-Arabinose （X）on the two points deliberately.
+iGEM14_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_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.
 == '''protocol''' ==