Team:ETH Zurich/blog
From 2014.igem.org
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Blog
- REDIRECT Team:ETH Zurich/labblog/20140829mod
Construction of the regulator plasmids
Thursday, July 5th
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 (1.0 kb). The vector piG0034 (pSEVA 181) was digested with the restriction enzymes HindIII and PacI to fiG0001 (3.0 kb). 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.
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 (0.8 kb). The backbone of the vector piG0040 (lasR in pSEVA 181) was amplified by PCR using oiG0007 and oiG0008. This fragment, fiG0011 (3.2 kb), 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 piG0041 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.
Construction of the rhlR regulator plasmid (piG0042)
Competent cells were transformed with piG0023 (C0171, rhlR) 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 rhlR by PCR using oiG0009 and oiG0010 resulted in the fragment fiG0006 (0.8 kb). The backbone of the vector piG0040 (lasR in pSEVA 181) was amplified by PCR using oiG0011 and oiG0012. This fragment, fiG0012 (3.2 kb), and fiG0006 were assembled using Gibbson assembly. Thus we formally replaced lasR by rhlR to construct the rhlR regulator plasmid piG0042. 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.
Construction of the luxR regulator plasmids with alternative constitutive promoters (piG0046 and piG0047)
The promoter site of the luxR regulator plasmid piG0041 was mutated by QuikChange site-specific mutagenesis to produce promoters of different strength. The primers oiG0031 and oiG0032 were used to establish piG0047, a plasmid with a promoter of intermediate strength (J23111). Analoguos the primers oiG0033 and oiG0034 were used to construct piG0046, a plasmid with a weak promoter (J23109). Competent cells were transformed with piG0046 and piG0047 and selected on ampicillin-LB-plates. The sequence was verified by Microsynth using the primers oig0035 and oiG0036.
Week 1 : Project selected
Wednesday, May 28th
After more than one month of endless meetings and passionate debates, we finally chose the project that will keep us occupied in the next five months. From the beautiful pattern made by the Sierpinski triangles, we will focus on cellular automata and try to implement one.
Sierpinski triangles appear when the rule 90 is followed by every cell on the grid :
Ideally we will use a microfluidic chip. We could also use a 3D printed agar plate like this one to load the colonies. On this grid we can implement the rule 6, which is the simplification of rule 90 considered as a rule with 2 inputs : each cell computes a simple XOR gate of its two parents.
The logic part will be built with integrases and the colony-to-colony communication will use quorum sensing.
Every colony will receive two quorum sensing signals (QSp and QSq) from the two cells above it. These two signals trigger the production of two different integrases r and s in the colony. Integrases enable to build biological XOR logic gates by switching twice a terminator. Indeed, every integrase can switch the terminator only once. Thus if the colony produces only r or only s, the terminator is switched only once, so the terminator is OFF, and GFP and QS1 or QS2 are produced (depending on the colony). If the colony produces r and s, the terminator is switched twice, so it is ON and it blocks expression of GFP and of the quorum sensing molecule.
We need to :
- find orthogonal quorum sensing molecules and orthogonal integrases
- discuss with microfluidics experts to check if using microfluidics is possible and presents advantages in our case
- find possible parts in the registry for integrases, and design plasmids
BSSE Openhouse Day
Saturday, May 10th
Sharing our iGEM and synthetic biology interest with the public
On May 10th the public in Basel had the unique chance to get an insight into many different scientific laboratories and the work done there. It was the joint open house day of D-BSSE of ETH (Department of Biosystems Science and Engineering) and the Biozentrum of the University of Basel. The many different labs opened their doors to the public and many scientists were present to give interested people some details about their daily work. So did the ETH iGEM team 2014. The team was present with a poster showing the history of iGEM, the previous ETH iGEM teams with their projects and general information about synthetic biology. Additionally there was a slideshow giving a best-of photo collection of last years jamboree. The goal of this day was to inform the public about synthetic biology in general and specifically about the spirit and the many different projects of iGEM. Many people showed strong interest in truly student driven projects and are curious to follow our team wiki for the next months.