Team:ETH Zurich/blog
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Integrase parameters
Digest and miniprep
Digest and miniprep
Miniprep
Preparation of DNA from iGEM kit
Sequencing, Transformation, antibiotic stock preparation
Miniprep
Project selected
BSSE Openhouse Day
- REDIRECT Team:ETH Zurich/labblog/20140829mod
Week 7: Human practice planning, Plasmid assembly running, Logo design
Wednesday, July 9th
- We have a logo !
- In the lab, we did some :
- Plasmid preparation
- Sequencing
- Digests
- Purification of backbone fragments needed for GA
- On the modeling side, we tried to estimate integrase parameters from the paper from Bonnet et al. [9], more particularly with the figure S4 :
We are trying several strategies : minimization of the error function and Markov Chain Monte Carlo.
- We have a precise plan for our human practice project. We would like to study the emergence of complexity in many different fields and investigate how people deal with it. We will do this with interviews of experts in different fields, and with a survey for a wider outreach. We will also reach younger people by organizing talks in schools. We will finally give our own advice on the question by writing an essay on the subject and linking it to our experience with Mosaicoli.
Digest and miniprep
Tuesday, July 8th
Optimizing Restriction Endonuclease Reactions
Digest of 291 and 561
291: (2x)
1μL AscI
1μL FseI
0.5μL PvuI
0.5μL AseI
20 μL DNA 291
5μL Cut Smart Buffer
fill up with 22 μL H2O to 50μL
dephosphorylation of 181 and 271, cleanup with PCR clean-up system, gel electrophoresis
Gel electrophoresis
0.5 g Agarose in 50 mL TAE (0.5x), heat in microwave to dissolve
let solution cool down to approximately 50 °C, add 5 μL peqGREEN, mix
pour solution in tray, use appropriate comb
add 10μL loading dye (6x) to samples
fill samples and ladder (10 μL) in wells
run gel at 135 V for 50min
Miniprep of 35 and 36
Digest and miniprep
Monday, July 7th
Optimizing Restriction Endonuclease Reactions
Digest of 291 and 561
291: (2x)
1μL AscI
1μL FseI
0.5μL PvuI
0.5μL AseI
20 μL DNA 291
5μL Cut Smart Buffer
fill up with 22 μL H2O to 50μL
Miniprep of 35 and 36
Miniprep
Sunday, July 6th
Miniprep with bacteria cultures containing 271, 291, 181 and 30
Addition of 200 μL Resuspension solution to culture pellet, vortex, addition of 200 μL Lysis solution
After ca. 5 min addition of 350 μL Neutralization solution, centrifuge samples for 10 min at 4000 rpm
Preparation of columns: addition of 500 μL Preparation solution, centrifugation for 1 min at 12 000 rcf, discard flow-through
Give supernatant of centrifuged samples onto columns, spin for 1 min at 12 000 rcf, discard flow-through
Addition of 500 μL Optional Wash Solution, spin for 1 min at 12 000 rcf, discard flow-through
Addition of 750 μL Wash Solution, spin for 1 min at 12 000 rcf, discard flow-through
Dry columns by centrifuging them for 2 min at 12 000 rcf, subsequently place columns in new collection tubes
Elute DNA with 100 μL Elution solution
According to Sigma-Aldrich Plasmid Miniprep Kit protocol
Preparation of DNA from iGEM kit
Friday, July 4th
E coli transformation
Addition of 10 μL H2O to appropriate well, wait for 5 min, transfer into sterile tube
Transformation of E. coli with pSEVA281 A-C2 (sfGFP), pSEVA271 C-A9 (mCherry) and piG0030:
Addition of 1 μL DNA to 75 μL competent cells (thawed on ice), put samples on ice for approximately 20 min
Heat shock: 90 s at 42 °C
Addition of SOC to samples, let cells recover for ca. 1h at 37 °C, 220 rpm
Plate 100 μL of bacteria suspension on LB-agar-plates containing the appropriate antibiotics (pSEVA281 A-C2, pSEVA271 C-A9: Kanamycin (50 μg/L), piG0030: Chloramphenicol (34 μg/L))
Let bacteria grow overnight at 37 °C
Sequencing, Transformation, antibiotic stock preparation
Thursday, July 3rd
Sequencing results
Biobricks of the plasmids piG0001, piG0015 and piG002 show the correct sequences
E coli Transformation
Transformation of E. coli with pSEVA181, pSEVA271, pSEVA291, pSEVA561.
Addition of 1 μL DNA to 75 μL competent cells (thawed on ice), put samples on ice for approximately 20 min
Heat shock: 90 s at 42 °C
Addition of SOC to samples, let cells recover for ca. 1h at 37 °C, 220 rpm
Plate 100 μL of bacteria suspension on LB-agar-plates containing the appropriate antibiotics (pSEVA181: Ampicillin (200 μg/L), pSEVA271 and pSEVA291: Kanamycin (50 μg/L), pSEVA561: Tetracycline (20 μg/L))
Let bacteria grow overnight at 37 °C
Antibiotic stock preparation
Preparation of antibiotics stock:
2 g Ampicillin were dissolved in 10 mL H2O and sterile filtered
0.5 g Kanamycin were dissolved in 10 mL H2O and sterile filtered
0.34 g Chloramphenicol were dissolved in 10 mL Ethanol
Week 6: Project planning, Gibson assemblies planning, First Matlab simulations
Wednesday, July 2nd
- We have a clear organization of the different experiments we would like to perform.
We want to optimize quorum sensing to have non-leaky non-cross-talking constructs. We want to prevent cross-talk between integrases and check their dynamics with different quorum sensing promoters. Then we will test the XOR gate without production of LuxI, to check how it works without the loop, and we will finally test our final construct with the loop, hoping that the delay between GFP production and the possible second switching due to AHL production is long enough to have enough fluorescence.
The red line is our critical timeline. Numbers of days are very optimistic,therefore we multiply the whole timeline by 2. The stars stand for modeling inputs.
- We have also clear plans for the Gibson assemblies to perform. As an example, here is the plan for regulator plasmids :
- We have a facebook page !
- We simulated quorum sensing without leakiness on Matlab, with parameters from the literature.
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.