Team:ETH Zurich/labblog/20140626

From 2014.igem.org

Revision as of 20:04, 22 July 2014 by Meistefa (Talk | contribs)

Contents

Construction of the regulator plasmids

Date : Thursday, 5th June (?)

Construction of the lasR regulator plasmid (piG0040)

Competent cells were transformed with piG0028 (K553003, lasR) and selected on chloramphenicol-LB-plates. Plasmid DNA was extracted by performing a miniprep. The relevant plasmid sequence was sequenced by Microsynth using the primers oiG0001 and oiG0002. Amplification of lasR by PCR using oiG0003 and oiG0004 resulted in the fragment fiG0004. The vector piG0034 (pSEVA 181) was digested with the restriction enzymes HindIII and PacI to fiG0001. The two fragments, fiG0001 and fiG0004, were assembled using Gibbson assembly. Thus we used the plasmid backbone of piG0034 and lasR of piG0028 to construct the lasR regulator plasmid piG0040. Competent cells were transformed with piG0040 and selected on ampicillin-LB-plates. Colony PCR using the primers oiG0035 and oiG0036 was conducted to check the size of the inserted fragment (expected bands at 1.0 and 1.2 kb). The sequence was verified by Microsynth using the same primers. F4.png PSEVA181.png 20140710 GA colony PCR.jpg

Construction of the luxR regulator plasmid (piG0041)

Competent cells were transformed with piG0008 (F2620, luxR) and selected on chloramphenicol-LB-plates. Plasmid DNA was extracted by performing a miniprep. The relevant plasmid sequence was sequenced by Microsynth using the primers oiG0001 and oiG0002. Amplification of luxR by PCR using oiG0005 and oiG0006 resulted in the fragment fiG0005. The backbone of the vector piG0040 (lasR in pSEVA 181) was amplified by PCR using oiG0007 and oiG0008. This fragment, fiG0011, and fiG0005 were assembled using Gibbson assembly. Thus we formally replaced lasR by luxR to construct the luxR regulator plasmid piG0041. Competent cells were transformed with piG0042 and selected on ampicillin-LB-plates. Colony PCR using the primers oiG0035 and oiG0036 was conducted to check the size of the inserted fragment (expected bands at 1.1 and 1.2 kb). The sequence was verified by Microsynth using the same primers. F11.png 20140712 piG0042 GA colony PCRs.jpg

Top