Difference between revisions of "Part:BBa K1412001"

Revision as of 09:38, 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

More information in linking page of the following pictures.

Characterization of backbone effect

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.

Characterization of IPTG effect

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.

Characterization of L-arabinose effect

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%.

Using semisolid culture medium to draw geometry pattern

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

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

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*]