Team:HKU-HKBU/speed control experiments
From 2009.igem.org
Contents |
Experiments
Plasmid extraction
5µl DH10B, containing the plasmid added into 5ml (anti-chloramphenicol) LB-Broth, 32 , overnight for plasmid extraction.
Experiment using MG3
Prepare competent cell: MG3 (delta CheZ strain)
Transformation: electroporation
Recover for 30 minutes
Culture with IPTG
Culture Time | IPTG concentration | |||
---|---|---|---|---|
0uM | 1uM | 2uM | 3.3uM | |
1hr | ||||
2hr | ||||
3hr | ||||
24hr |
Western Blotting
Equal loading
2ul sample -> 96 well plate -> add 100ul BCA solution (50:1). After measurement at wavelength of 562nm, total amount of protein = sample concentration x sample volume. Adjust the sample volume to the same loading volume by adding the mixture buffer.
Result
According to the results from western Blotting, there is no obvious positive relation between the cheZ expressing and the concentration gradient of IPTG, in the range from 0 to 3.3uM in the first three hours . Above is the expressing of cheZ overnight, 0, 1,2 and 3.3 uM from left to right respectively. The positive relationship is much more obvious after 24 hours.
Swimming test
Test MG3-plD-his-cheZ-cm under 0.0005, 0.001, 0.002, 0.004, 0.008, 0.012mmol/L IPTG.
No Chloramphenicol
2nd time: Chloramphenicol added
Analysis
- Difference between various IPTG conc. gradients not significant. The expressing level of cheZ under various concentration gradients of IPTG is not significantly different within 8 hours, leading to the similarity of swim behaviors.
- Even negative control (no IPTG added) swims, probably due to leak-expressing.
- The speed enters plateau after 4 hours, about 2.25-2.5mm/hr.
Experiment using E.Coli 2443
cheZ knock-out: Refer to QQ
Co-transformation: Failure