http://2013.igem.org/wiki/index.php?title=Special:Contributions/Gabyqz&feed=atom&limit=50&target=Gabyqz&year=&month=2013.igem.org - User contributions [en]2024-03-28T15:58:56ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:BIOSINT_Mexico/SensorTeam:BIOSINT Mexico/Sensor2013-09-28T04:21:39Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Sensor</h1> </html><br />
----<br />
----<br />
===Xenobiotics Sensor=== <br />
<br />
[[File:Plaguicidas.jpg|470px|right]]<br />
<br />
:The sensor consists in the application of the model development by Debrecen in 2010, using nuclear receptors as transcriptional factors1 . The family of nuclear receptors are composed by a series of proteins which acts like sensor for lipids, steroids, hormones,endocrine-disrupting chemicals, etc.2<br />
<br />
:Several xenobiotic substances are present in the intestine and many of them result toxic for the host. One example is organochlorine pesticide, a endocrine-disrupting chemical, which is a molecule that can be found on contaminated food and represent an important toxicity source. Detection and decrease levels of pesticide could be an effective way to solve the problem .3<br />
<br />
:Nuclear receptors are ligand specific, and are composed by two basis domains: ligand binding domain (LBD) and DNA binding domain (DBD).2 In our sensor we adapt and characterized Debrecen system, which fuses specific LBD with Gal 4 DBD. 1<br />
<br />
:The system adapt TRE-CMV promoter; that has a basal expression in the absence of TetR, Gal 4; yeast transcription factor galactose 4, PXR ligand binding domain which binds organochlorine and PolyA; important for nuclear export. The complete system will be expressed in one plasmid. 1<br />
<br />
:Finally the nuclear receptor will interact with UAS, using Gal4 as specific binding region. UAS and Gal-4 forms a complex which activate the expression of the L.plantarum promoter. Development of specific nuclear receptors helps to quantify accurately the expression of the report product in relation with the ligand concentration. 4<br />
<br />
:The probiotic sensor detect organochlorine, then the nuclear receptor complex activates the promoter for the expression of linA enzyme, which catalyze the conversion of γ-hexachlorocyclohexane to 1,2,4-trichloro benzene.5Using this method our probiotic is not only available to detect organochlorine pesticides but also degrade capabilities of the molecule, contributed to decrease the presence of the toxic molecule .<br />
<br />
<br />
[[File:Detection_.jpg|center|300px]]<br />
<br />
[[File:Sensorxeno.jpg|center|400px]]<br />
<br />
<br />
===REFERENCES===<br />
<br />
----<br />
<br />
<br />
1.iGEM Debrecen (2010).Recovered from https://2010.igem.org/Team:Debrecen-Hungary/favorite<br />
<br />
2. Willson, T. et al.(2002) PXR, CAR and drug metabolism. Nature Publishing group: Volume 1<br />
<br />
3.Kojima, H, et al. (2010). Endocrine- disrupting potential of pesticides via nuclear receptors and aryl hidrocarbon receptor. Journal of health science. <br />
<br />
4. Elliot, D. & Brand A. (2002) The Gal4 system. Methods in molecular biology in Drosophila: A Fly Geneticist's Swiss Army Knife.<br />
<br />
5.Yang, J, (2011). Construction of a Genetically Engineered Microorganism that Simultaneously Degrades Organochlorine and Organophosphate Pesticides. Appl Biochem Biotechnol.</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ChassisTeam:BIOSINT Mexico/Chassis2013-09-28T04:17:16Z<p>Gabyqz: /* Backbones registry testing */</p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
__NOTOC__<br />
<br />
----<br />
<html><h1 class="myowntitle">Chassis</h1> </html><br />
----<br />
----<br />
===Lactobacillus chassis===<br />
----<br />
<br />
<br />
<br />
<br />
:This year the Biosint Mexico team will develope a smart probiotic. Throughout the competition several projects about probiotics have been present, nevertheless the main disadvantage was that most of them were not being created in a lactobacillus strain. Because of this we constructed a Lactobacillus platform for others iGEM teams. <br />
<br />
[[File:Backbonevec.jpg|center|300px]]<br />
<br />
<br />
=== L. plantarum?=== <br />
----<br />
<br />
<br />
<br />
[[File:Plantarum.jpg|right|300px]]<br />
<br />
:Lactobacillus plantarum is a gram-positive lactic acid bacterium, present naturally in dairy products, as well as in the gastrointestinal tract1. <br />
<br />
:We chose to use L. plantarum as a chassis , because of its unique ability to grow and adapt to a large number of niches2. Studies have shown that L. plantarum has the capacity to optimize its genetic material in order to adapt to different environments, especially those with high levels of carbohydrates2. Another special property of L. plantarum is that it has a high tolerance towards acids, and it is present in many fermentative processes2.<br />
<br />
:These characteristics among others make it a potential candidate for its use in different industries. Using recombinant DNA technology, this bacterium could be applied to food industries as a starter or marker in fermentative processes, in healthcare industry as a probiotic or in biofuel production2. As part of our project, we will be explore the potential of L. plantarum as a probiotic, and produce biobricks that can join all the beneficial properties of different probiotics and express it in our own chassis.<br />
<br />
===Backbones registry testing===<br />
----<br />
<br />
:Before we tried to design a backbone compatible with the biobricks registry, we make experiments in order to know if some backbones in the registry was already compatible with a lactobacillus chassis. <br />
<br />
:Tests for psB4K5, psB3K5, psB2K3, psB1A7 was made it. Unfortunatley we are not avaible to transform lactobacillus plantarum with any of the plasmids.<br />
<br />
=== Vector design ===<br />
----<br />
<br />
[[File:backboneplant.jpg|left|200px]]<br />
<br />
<br />
:Using psb1c3 as template we design a new backbone compatible with E.coli and lactobacilli. In our case we used the bases of a staphylococcal plasmid, pUB110, that replicates by a rolling-circle mechanism via a single-stranded (ss) DNA intermediate in Bacillus subtilis and Staphylococcus aureus, and this mechanism is widespread among gram-positive bacteria . When pUB110 replicates in cells of B.subtilis or S. aureus, the plasmid regions, rep, ori, and BA3, function as a structural gene that encodes a replication protein.<br />
<br />
:The BA3 minus origin has the ability to increase stability of the plasmid in its active host (7, 33), effects of palA and the IG region on plasmid stability were examined in L. casei MSK248.. These results indicate that palA and the IG region of M13 provide increased stability to the plasmids in which they have been incorporated in L. casei.<br />
<br />
:We used the minus origin mention before and paste into psb1c3 in order to replaced the origin of replication that the vector actually have. We also add GFP to the plasmid, to measure transformation efficiency.<br />
<br />
<br />
<br />
===REFERENCES:=== <br />
----<br />
<br />
1Lee, J., Halgerson, J., Kim, J., and O’Sullivan, D. (2007). Comparative Sequence Analysis of Plasmids from Lactobacillus delbrueckii and Construction of a Shuttle Cloning Vector. [Online]. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1932812/ on April 16, 2013.<br />
<br />
2Siezen, R. and Hylckama, J. (2011). Genomic diversity and versatility of Lactobacillus plantarum, a natural metabolic engineer. [Online]. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3271238/ on April 16, 2013.</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/AcknowledgmentTeam:BIOSINT Mexico/Acknowledgment2013-09-28T04:16:25Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Special Thanks</h1> </html><br />
----<br />
----<br />
<br />
[[File:futureigem.jpg|800px|center]]<br />
<br />
:We would like to give our most significative thanks to all the people that supported and helped us. Specially thanks to the people that work in our university, '''Instituto Tecnologico de Estudios Superiores de Monterrey Campus Queretaro''', because of them we are right where we are now. At this moment, it is very likely that we are not be able to say whatever we want to them, despite this, we are very happy with their own contributions to our project.<br />
<br />
:We have to recognize that, specially this people, brought us answers that we were not able to find by ourselves. For us, for our team, and for our proyect, the most important thing they did was their support that came together with very useful advices. <br />
<br />
:Our special thanks are principally to:<br />
<br />
'''-Dra. Sonia Vazquez Flores''' One of our principal advisors and encouragers to iniciate our team and project.<br />
<br />
'''-Ing. Rodrigo Machado''' Our lastest president and now recently graduated, and the most important thing, our friend who always has been there for supporting us. <br />
<br />
'''-Ing. David Maycotte''' Ex member of this team which we appreciate for his unconditional support.<br />
<br />
'''-Ing. Antonio Villarreal ''' Ex member of our team, actually now he is studying a mastery on Synthetic Biology in Paris, France. He helped a lot with the building of our construct.<br />
<br />
'''-Aldo Diaz''' Ex member of this team, and our friend. We appreciate that he always helped in every problem we told him.<br />
<br />
'''-Citlalli Rosas''' Director of "Grupos Estudiantiles" and principal advisor in our bureocratic procedures.<br />
<br />
'''-Dr. Gerardo Montejano''' Director of Food Industry, Biotechnology and Agronomist Engineering School. He always has been supporting us in whatever we need related with the Department.<br />
<br />
'''-Dr. Hector Morelos''' Director of Engineering and Architecture Division. He, just like Dr. Morelos did, supported and helped our project.<br />
<br />
'''-Dr. Alejandro Olmos''' He teaches Enzimology and Biocatalisis. He did very significant things for us, just like giving us very worth advises and also he could acquire L. Plantarum for our project.<br />
<br />
'''-Dr. Javier Rangel''' Molecular Biology and Biosensors teacher, that helped us at the begginig of our project solving some questions and bringing us ideas.<br />
<br />
'''-Ambey Shree Shukla''' Molecular Diagnostic and Functional Genomics teacher. She is a very polite person, always gentle with the team. She also always showed respect and interest for helping in the project.<br />
<br />
'''-Prashant Kumar Mishra''' Ex professor of Genetic Engineering. He was one of our first advisors, and no matter what, where, and when (even 2:00 a.m.) he was always for whatever we needed.</div>Gabyqzhttp://2013.igem.org/File:Futureigem.jpgFile:Futureigem.jpg2013-09-28T04:15:53Z<p>Gabyqz: </p>
<hr />
<div></div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_MexicoTeam:BIOSINT Mexico2013-09-28T04:12:12Z<p>Gabyqz: /* Project Description */</p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">HOME</h1> </html><br />
----<br />
----<br />
<br />
=== Project Description ===<br />
<br />
[[File:Fac1.jpg|800px|center]]<br />
<br />
<br />
<br />
:As the omnipotent Tezcatlipoca (lord of earth and heaven in the Aztec mythology) we also wanted to create something special, so we were thinking what can we do using synthetic biology and then out of nothing we had an epiphany and got the idea for the iGEM competition. This year Biosint Mexico team will develop a smart probiotic. Even though Cinteotl (lord of the corn in the Aztec mythology) had been insisting us on doing something with corn and Tlaloc (lord of rain in the Aztec mythology) agreed with him promising us good rain if we had to raise crops, but still we decided to go for the probiotic.<br />
<br />
<br />
:Along the competition have been presented several projects about probiotics, nevertheless the main disadvantage is that most of them were not being created in a Lactobacillus strain. Because of this we will construct a Lactobacillus platform for others iGEM teams.<br />
<br />
<br />
:This time Quetzalcoatl helped us out giving us the idea of our pet that was used for the previous iGEM team, and this year is also representing our team as the official pet.<br />
:Continuing with the idea of the smart probiotic system we will also include a sensor for xenobiotic substances that will detect and decrease intoxications by pesticides. As the mighty cougar that rules the jungle, this smart probiotic will provide a possible solution for some health issues in the Mexican camp.<br />
<br />
<br />
[[File:LogotipoDefinitivo.png|800px|center]]</div>Gabyqzhttp://2013.igem.org/File:GFP2.jpgFile:GFP2.jpg2013-09-28T04:07:24Z<p>Gabyqz: </p>
<hr />
<div></div>Gabyqzhttp://2013.igem.org/Team:BIOINT_Mexico/Lab_workTeam:BIOINT Mexico/Lab work2013-09-28T04:05:33Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Lab work</h1> </html><br />
----<br />
----<br />
<br />
'''Experiment 1: Preparation of growth mediums (MRS and LB) (June 5th, 2013)'''<br />
<br />
10 Agar LB plates<br />
<br />
15 Agar MRS plates<br />
<br />
<br />
Notes:<br />
*(1) 18.6g of Agar MRS were weighted to prepare 300ml of medium<br />
*(2) 7g of Agar LB were weighted to prepare 200ml of medium<br />
*(1) 5g of A-MRS were diluted in 80ml of distilled water<br />
*Another 5g and 170ml of distilled water were added<br />
*Small lumps were formed in the bottom of the Erlenmeyer flask<br />
*(2) 7g of Agar LB were diluted in 100ml of distilled water<br />
*Further 100ml of distilled water were added<br />
*It wasn’t possible to use a control of the sterilization because there was no autoclave tape<br />
*The autoclave began heating up<br />
*The autoclave valve was lowered<br />
*When the temperature of the autoclave reached 120⁰c, the power was cut to the half<br />
*The vapor cycle ended and the temperature decreased<br />
*The temperature reached 105⁰c and the valve was opened. The mediums were left inside the autoclave until de laminar flow cabinet was unoccupied<br />
*The medium in petri dishes were stored in the refrigerator <br />
*The growth mediums in storage were revised, they do not show signs of contamination <br />
17 MRS Agar<br />
<br />
9 LB Agar<br />
<br />
<br />
----<br />
'''Experiment 2: Preparation of MRS broth and glycerol stocks (July 8th, 2013)'''<br />
<br />
*0.7679g of powder for MRS broth were weighted<br />
*15ml of distilled water were measured in a 100ml measuring cylinder <br />
*Besides, 200ml were prepared for future use<br />
*10.009g of powder for MRS broth were weighted<br />
*It was diluted in 100ml of distilled water<br />
*Another 100ml of distilled water were added<br />
*0.7679g of powder for MRS broth were diluted in 15ml of distilled water<br />
*Both flasks were marked with autoclave tape and they were sterilized for 15 minutes at 121⁰c<br />
*They cooled down and the autoclave tape confirmed the sterilization<br />
*The 200ml broth was stored properly labeled <br />
*The 15ml of MRS broth and the 15ml of glycerol were mixed to 100% in the laminar flow cabinet. They were gently shaken until a homogenous mixture was obtained in a falcon tube of 45ml<br />
*An aliquot of L. Plantarum was added to the broth and glycerol solution and it was sealed with parafilm<br />
*It was left incubating at 37⁰c and 200rpm<br />
*After 24 hours the sample was taken out of the incubator to striate it<br />
*2500 (x2)ml were placed in 22 eppendorf tubes using a micropipette of 200µl to cultivate and recompile the mixture of L. Plantarum contained in the falcon tube <br />
*1 eppendorf tube was centrifuged at 13.4 rpm for 3 minutes <br />
*After the centrifugation there was a small pellet left, from which the supernatant was discarded and a solution of MRS broth (500ml) was added <br />
*Once the mixture was made, two petri dishes with MRS Agar were striated in the laminar flow cabinet. A third dish was striated in case of an emergency<br />
*The petri dishes were placed facing down in the incubator at 37⁰c and 200 rpm<br />
*The remaining samples (21 falcon tubes) were stored in deep freezing <br />
*E. Coli K12 was striated in two petri dishes with LB Agar <br />
*They were placed in the incubator at 37⁰c.<br />
<br />
<br />
----<br />
'''Experiment 3: Plasmid purification (July 16th, 2013)'''<br />
<br />
*An eppendorf tube of 1.5 ml with L. Plantarum culture was centrifuged at 12000 rpm for 1 minute two times<br />
*The supernatant was discarded and the tube’s content was resuspended with more L. Plantarum culture in MRS broth<br />
*The eppendorf tube was centrifuged for 15min at 6000 rpm<br />
*The supernatant was discarded <br />
*The pellet was resuspended in 250 µl resuspension buffer<br />
*250 µl of Lysis Buffer L7<br />
*The tube was gently mixed inverting it 5 times carefully<br />
*350 µl of precipitation Buffer N4 were added and mixed softly inverting the tube<br />
*It was centrifuged at 12000 rpm for 10 minutes<br />
*The supernatant was transferred to a spin column inside a washtube <br />
*It was centrifuged at 12000 rpm for a minute<br />
*The supernatant was discarded and 500 µl of Wash Buffer with ethanol (w10) were added to the column<br />
*It was incubated for one minute at room temperature<br />
*The column was centrifuged at 12000 rpm for 1 minute<br />
*The liquid from the washtube was discarded and the column was placed inside the tube<br />
*700 µl of Wash Buffer W9 with ethanol were added to the column<br />
*The column with the washtube was centrifuged at 12000 rpm for 1 minute<br />
*The liquid from the washtube was discarded<br />
*The column in the washtube was centrifuged at 12000 rpm for 1 minute<br />
*The liquid from the washtube was discarded<br />
*The column was placed inside an eppendorf tube of 1.5ml<br />
*75 µl of preheated TE Buffer were added at the center of the column<br />
*The TE Buffer was previously warmed in water bath at 65⁰c-70⁰c for 3 minutes<br />
*The column was incubated for 1 minute at room temperature<br />
*The column was centrifuged at 12000 rpm for 2 minutes<br />
*The eppendorf tube contains the purified plasmid<br />
<br />
<br />
----<br />
'''Experiment 4: Preparation of mediums for lactobacillus and E. Coli (July 17th, 2013)'''<br />
<br />
*15.3 g of MRS were weighted for 300 ml, the 2% (0.306 g.) of the MRS was used to do agar-agar<br />
*The autoclave started to heat <br />
*8.75 g of LB agar were weighted to dilute in 500 ml of distilled water<br />
*0.5005 g of LB broth were weighted to be diluted in 250 ml of distilled water<br />
*The 3 mediums were introduced in the autoclave<br />
*After 54 minutes the sterilization was complete <br />
*0.750 g of Kanamycin sulfate were measured and they were diluted in 15 ml of distilled water<br />
*1.5 g of Ampicillium sodium were weighted and diluted in 15 ml<br />
*The mediums were collocated in the laminar flow cabinet to pour into the petri dishes and striate <br />
*The mediums never solidified <br />
*2% more of agar was added in the LB broth and the mediums were placed in the autoclave <br />
*The mediums were collocated in the laminar flow cabinet to be poured into the petri dishes and striate<br />
*But the mediums didn’t solidified<br />
<br />
<br />
----<br />
'''Experiment 5: Preparation of MRS agar mediums (July 19th, 2013)'''<br />
<br />
*The mediums didn’t solidify, because the agar added wasn’t enough<br />
*10.2 g of MRS broth were measured and 2 g of agar-agar were added<br />
*The pH of the mediums was 6.6 <br />
*A stock of HCl at 10% was added until the pH reached 6.4 <br />
*The mediums were sterilized with the autoclave for 15 minutes at 121°C <br />
*40 ml of agar were mixed with 40 µl of stock of ampicillin and they were poured into 2 petri dishes<br />
*40 ml of MRS agar were mixed with 40 µl of kanamycin stock and they were poured into 2 petri dishes <br />
*6 petri dishes were made with only MRS agar <br />
*2 control petri dishes with MRS agar were striated with L. Plantarum with 1.5 ml of glycerol stock <br />
*The petri dishes were incubated at 37°C for 2 hours<br />
<br />
<br />
----<br />
'''Experiment 6: Growth of E. Coli (July 21st, 2013)'''<br />
<br />
*No growth was discernible in the petri dishes with E. Coli, which were left incubating. This attributed to the use of an E. Coli sample, which was in refrigeration since 2011<br />
*The LB Agar medium was heated in an electric stove for approximately 10 minutes<br />
*A new sample of lyophilized E. Coli sample was opened and a representative sample was extracted to be rehydrated with LB broth<br />
*Liquid LB agar was poured into 2 petri dishes and they were left to solidify<br />
*Both dishes were striated with the rehydrated E. Coli<br />
*Both dishes were left incubating, after being sealed, during 24 hours at 37⁰c<br />
<br />
<br />
----<br />
'''Experiment 7: Glycerol stocks reactivation (July 22nd, 2013)'''<br />
<br />
*No discernible growth was observed in the L. Plantarum plates after a 72 hours incubation period. After consulting an external source, it was determined that this was due to glycerol stocks used, which weren’t reactivated<br />
*Two stocks of glycerol were reactivated with MRS broth<br />
*10 ml of MRS broth were added to a 1 ml stock in a falcon tube<br />
*The second stock was poured into another falcon tube, the result was 1.5 ml of stock with 15 ml of MRS broth<br />
*Both falcon tubes were sealed and left incubating at 37⁰c<br />
<br />
<br />
----<br />
'''Experiment 8: Overnight growth of E. Coli in LB broth (July 23rd, 2013)''' <br />
<br />
*5 ml of LB broth were poured into 2 falcon tubes each <br />
*An E. Coli colony was added to each tube with broth <br />
*Both tubes were sealed and left incubating at 37⁰c<br />
<br />
<br />
----<br />
'''Experiment 9: Preparation of mediums for lactobacillus and E. Coli (July 24th, 2013)'''<br />
<br />
*15.3 g of MRS and 6 g of bacto agar were weighted to be diluted in 300 ml<br />
*17.5 g of LB agar were weighted to prepare 500 ml<br />
*The MRS and LB agar were autoclaved <br />
*1 ml of E. Coli was inoculated in 100 ml of LB agar in a flask of 250 ml<br />
*5 g of LB broth were weighted<br />
*It was autoclaved for 15 minutes at 121⁰c<br />
<br />
<br />
----<br />
'''Experiment 10: Preparation of TE buffer (July 26th, 2013)'''<br />
<br />
*6.05 g of TRIS were weighted and diluted in 60 ml of distilled water<br />
*9.3041 g of EDTA were weighted and diluted in 60 ml of distilled water<br />
*The TRIS’ pH was 10.29, therefore HCl was added until the pH reached 8.3<br />
*The EDTA’s pH was 3.25, therefore NaOH crystals were added until the pH reached 7.79<br />
*They were sealed and autoclaved for 15 minutes at 121⁰c<br />
<br />
<br />
----<br />
'''Experiment 11: Preparation of competent E. Coli cells (July 28th, 2013)'''<br />
<br />
*A falcon tube was prepared with 50 ml of CaCl2 0.1M<br />
*Another falcon tube was prepared with CaCl2 0.1M/ 15% glycerol<br />
*4 eppendorf tubes of 1.5 ml were filled <br />
*The 4 tubes were left in ice for 10 minutes<br />
*The 4 tubes were centrifuged for 3 minutes at 6000 rpm and the supernatant was discarded<br />
*1.5 ml of the aforementioned culture were added to each tube<br />
*The tubes were centrifuged for 3 minutes at 6000 rpm <br />
*The supernatant was discarded<br />
*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M for each tube<br />
*The 4 tubes were incubated in ice for 20 minutes<br />
*The tubes were centrifuged again for 3 minutes at 6000 rpm<br />
*The supernatant was discarded<br />
*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each<br />
*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c<br />
<br />
<br />
----<br />
'''Experiment 12: L. Plantarum plasmid purification (July 28th, 2013)'''<br />
<br />
*1.5 ml of L. Plantarum culture in MRS broth were poured into 2 eppendorf tubes, 1.5 ml each<br />
*Both tubes were centrifuged for 4 minutes at 12000 rpm<br />
*The supernatant was discarded and another 1.5 ml of culture were added <br />
*Both tubes were centrifuged for 4 minutes at 12000 rpm<br />
*The supernatant was discarded <br />
*250 µl of resuspension buffer (R3) were added and the pellets were resuspended until the mixture was homogenous <br />
*The tubes were incubated at room temperature for 15 minutes<br />
*250 µl of Lysis buffer (L7) were added to each tube. They were gently mixed<br />
*The tubes were incubated at room temperature for 5 minutes<br />
*350 µl of precipitation buffer (N4) were added <br />
*Both tubes were vigorously mixed until a homogenous mixture was obtained <br />
*The tubes were centrifuged at 12000 rpm for 10 minutes<br />
*The supernatant was poured into a spin column in a wash tube of 2 ml <br />
*The columns were centrifuged at 12000 rpm for 1 minute <br />
*The supernatant was discarded and the column was placed in the new washtube<br />
*500 µl of wash buffer (W10) with ethanol were added to the column <br />
*The columns were incubated for 1 minute at room temperature<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The liquid that went through the columns was discarded<br />
*700 µl of wash buffer (W9) were added<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The liquid that went through the columns was discarded<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The liquid that went through the columns and the washtube was discarded<br />
*The columns were placed in eppendorf tubes of 1.5 ml<br />
*70 µl of TE buffer (previously heated at 65⁰c for 3 minutes) were added in the center of each column<br />
*The columns were incubated at room temperature for 1 minute <br />
*The columns were centrifuged at 12000 rpm for 2 minutes<br />
*The eppendorf tubes contain the purified plasmids<br />
*The columns were removed, the eppendorf tubes were sealed and they were refrigerated at 4⁰c until the next day<br />
<br />
<br />
----<br />
'''Experiment 13: Electrophoresis gel for L. Plantarum (July 29th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution <br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
<br />
Preparation of the sample to be placed in the gel<br />
*10 µl of each sample of purified plasmid were placed in eppendorf tubes of 1.5 ml<br />
*5 µl of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample<br />
<br />
Running the gel<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*15 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 15 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*No sample presented marks in the gel, only the gene ruler<br />
<br />
<br />
----<br />
'''Experiment 14: Overnight culture of L. Plnatarum and agarose preparation (July 29th, 2013)'''<br />
<br />
*2 colonies with one day of incubation were inoculated in MRS broth out of a 10 ml sample<br />
*They were placed in falcon tubes and left incubating at 37.2⁰c<br />
*35 ml of a TAE 1x solution were used to prepare agarose gel for electrophoresis. 0.35 g of agarose were added to the solution to solidify it<br />
*The liquid gel was poured into a casting tray and the well combs were placed<br />
<br />
<br />
----<br />
'''Experiment 15: Rehydration of plasmids in kit plates (July 30th, 2013)'''<br />
<br />
*The plate kits were numbered as indicated in the iGEM’s parts registry and the plasmids to be used were localized<br />
*The protective layer was pierced with a micro-pipet<br />
*Each plasmid was rehydrated with 10 µl of distilled water<br />
*The liquid was pipetted until it was homogeneous <br />
*It was left resting for 5 minutes to ensure its rehydration<br />
<br />
<br />
----<br />
'''Experiment 16: Electrophoresis gel for kit plates plasmids (July 30th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to form a 400 ml solution<br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
*The agarose with TAE 1x was heated in the microwave for 4 seconds<br />
Preparation of the sample to be run in gel<br />
*5 µl of each rehydrated plasmid were placed in eppendorf tubes <br />
*10 µl of 6x DNA Loading Dye were added and it was pipetted until the mixture was homogeneous<br />
Running the gel<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*15 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 15 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*Thin marks can be appreciated in samples psB4A5 and psB1A7<br />
<br />
<br />
----<br />
'''Experiment 17: E. Coli transformation with linearized plasmids (psB4k5, psB1k3, psB2k3) (July 31st, 2013)'''<br />
<br />
*The competent cells were taken out of deep freeze and were left to defrost in ice<br />
*They were mixed softly and 50 µl of competent cells were placed in a cold eppendorf tube <br />
*10 µl of linearized plasmid were added (psB4k5)<br />
*It was mixed gently and left incubating in ice for 30 minutes<br />
*The tube was put through heat shock in water at 42⁰c for 45 seconds<br />
*The tube was placed in ice for 2-5 minutes<br />
*900 µl of S.O.C. were added<br />
*The tube was left incubating for 1 hour at 37⁰c and 200 rpm<br />
*A dish with LB agar and kanamycin was striated with the transformed E. Coli<br />
*The dish was sealed and left incubating at 37⁰c<br />
*The same procedure was followed for plasmids psB1k3 and psB2k3<br />
<br />
<br />
----<br />
'''Experiment 18: Preparation of transformation buffer CaCl2 50mM (August 2nd, 2013)'''<br />
<br />
*1 ml of CaCl2 buffer 200mM were placed in a beaker<br />
*3 ml of sterile distilled water were added<br />
*With a syringe, it was filled to its maximum capacity with the buffer<br />
*A ministart 0.2µm filter was connected to the syringe<br />
*The content of the syringe was poured into a sterile falcon tube through the tube <br />
*The tube was left cooling in ice<br />
<br />
<br />
----<br />
'''Experiment 19: E. Coli transformation with pGLO (GFP) (August 3rd, 2013)'''<br />
<br />
*The competent cells were taken out of deep freeze and left defrosting in ice<br />
*They were gently mixed and 50 µl were placed in a cold 1.5 ml eppendorf tube<br />
*10 µl of pGLO were added<br />
*They were gently mixed and left incubating in ice for 30 minutes<br />
*The tube went through heat shock in water at 42⁰c for 45 seconds<br />
*The tube was left in ice for 5 minutes<br />
*900 µl of S.O.C. was added to the tube<br />
*It was gently mixed and left incubating for 1 hour at 37⁰c and 150 rpm <br />
*A control dish and a dish with antibiotic were striated <br />
*Both dishes were sealed and left incubating facing down at 37⁰c<br />
<br />
<br />
----<br />
'''Experiment 20: MRS and LB mediums preparation (August 5th, 2013)'''<br />
<br />
*15.3 g of MRS and 6 g of bacto agar were weighted to be diluted in 300 ml<br />
*17.5 g of LB agar were weighted to prepare 500 ml<br />
*The MRS and LB agar were autoclaved for 15 minutes at 121⁰c<br />
*They were left in refrigeration<br />
Results:<br />
*After an incubating period of 24 hours both, the control and the L-arabinose and ampicillin, plates were observed. Both plates presented bacterial growth. The bacteria in the L-arabinose plate were analyzed in the transilluminator and they glowed. Therefore, the transformation was successful and the gen was expressed correctly. Likewise, it was demonstrated that the previously prepared competent cells are effective. <br />
<br />
<br />
----<br />
'''Experiment 21: Preparation of plates with LB agar and antibiotic (August 6th, 2013)'''<br />
<br />
*The LB agar was heated in the electrical stove until it was completely liquefied <br />
*The next plates were prepared:<br />
**1 control (only agar)<br />
**2 with kanamycin<br />
**1 with kanamycin and L-arabinose<br />
**1 with ampicillin<br />
*Plate with kanamycin<br />
**40 µl of kanamycin and 40 ml of LB agar were poured into a beaker (for 2 dishes)<br />
**They were mixed and poured into 2 dishes<br />
*Plate with kanamycin and L-arabinose<br />
**20 µl of kanamycin, 600 µl of L-arabinose and 20 ml of LB agar were mixed in a beaker<br />
**The solution was poured into a petri dish<br />
*Plate with ampicillin<br />
**20 µl of ampicillin and 20 ml of agar were mixed in a beaker<br />
**The solution was poured into a dish<br />
*The mediums were prepared again to duplicate the amount of dishes<br />
*The plates solidified and they were properly labeled<br />
*A total of 9 dishes were prepared<br />
**1 with LB agar<br />
**4 with kanamycin<br />
**2 with kanamycin and L-arabinose<br />
**2 with ampicillin<br />
<br />
<br />
----<br />
'''Experiment 22: E. Coli transformation with linearized plasmids (psB4k5, psB1A7, psB2k3) (August 7th, 2013)'''<br />
<br />
*The DNA of each plasmid was rehydrated with 10 µl of sterile distilled water<br />
*Each well of the kit plates was pierced with a micro pipet<br />
*Each rehydrated DNA was stored in eppendorf tubes<br />
*The competent cells were taken out of deep freeze and were left to defrost in ice<br />
*The competent cells were mixed softly and 50 µl of competent cells were placed in a cold eppendorf tube <br />
*2 µl of linearized plasmid were added and pipetted until it was homogeneous <br />
*It was sealed and left incubating in ice for 30 minutes<br />
*The tube was put through heat shock in water at 42⁰c for 60 seconds<br />
*The tube was placed in ice for 5 minutes<br />
*200 µl of S.O.C. were added<br />
*The tube was left incubating for 2 hours at 37⁰c<br />
*(This was done with each plasmid)<br />
*The samples were striated with a Digralsky spreader in dishes with their correspondent antibiotic and/or inducer<br />
*For each plasmid it was inoculated:<br />
##In a dish, 20µl of a sample of transformed E. Coli (with antibiotics)<br />
##In a dish, 100 µl of the same transformed E. Coli (with antibiotics)<br />
##In a control dish, the rest of the transformed cells<br />
*they were sealed and left incubating at 37⁰c<br />
<br />
<br />
----<br />
'''Experiment 23: Preparation of competent cells with Lactobacillus Plantarum (August 8th, 2013)'''<br />
<br />
*2 eppendorf tubes of 1.5 ml were filled with L. Plantarum in MRS broth<br />
*The tubes were left in ice for 10 minutes<br />
*The tubes were centrifuged for 3 minutes at 6000 rpm and the supernatant was discarded<br />
*1.5 ml of the aforementioned culture were added to each tube<br />
*The tubes were centrifuged for 3 minutes at 6000 rpm <br />
*The supernatant was discarded<br />
*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M for each tube<br />
*The tubes were incubated in ice for 20 minutes<br />
*The tubes were centrifuged again for 3 minutes at 6000 rpm<br />
*The supernatant was discarded<br />
*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each<br />
*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c<br />
<br />
<br />
----<br />
'''Experiment 24: Purification of L. Plantarum plasmids (August 9th, 2013)'''<br />
<br />
*2 different samples of L. Plantarum were used, one from an isolated colony and another form a reactivated stock<br />
*1.5 ml of each sample were poured into eppendorf tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*Another 1.5 ml of the samples were added to the tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixtures were homogenous <br />
*The tubes were incubated for 15 minutes<br />
*250 µl of Lysis buffer (L7) were added to each tube and mixed gently<br />
*They were left incubating for 5 minutes<br />
*350 µl of precipitation buffer were added and it was vigorously shaken<br />
*Both tubes were centrifuged at 12000 rpm for 10 minutes<br />
*It was observed that before centrifuging the samples, the tube with the reactivated cells had a much larger volume than the other, which meant a mistake was probably made when pipetting <br />
*The supernatant was transferred to a spin column in a 2 ml washtube <br />
*The tubes were centrifuged for 1 minute at 12000 rpm<br />
*The liquid that passed through was discarded and the column was placed again in the washtube<br />
*500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute<br />
*Both tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded<br />
*700 µl of Wash buffer (W9) were added<br />
*Both columns were centrifuged at 12000 rpm for 1 minute<br />
*The supernatant was discarded and the columns were transferred to eppendorf tubes of 1.5 ml <br />
*70 µl of TE buffer were added to each tube (previously heated at 65⁰c for 3 minutes)<br />
*The tubes were incubated for 1 minute at room temperature<br />
*Both tubes were centrifuged at 12000 rpm for 2 minutes<br />
*The columns were discarded and the tubes were sealed and refrigerated at 4⁰c<br />
<br />
<br />
----<br />
'''Experiment 25: Gel for electrophoresis of plasmids (August 9th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution <br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
*The agarose with the gel was heated in the microwave for 45 seconds<br />
Preparation of the sample to be placed in the gel<br />
*10 µl of each sample of purified plasmid were placed in eppendorf tubes of 1.5 ml<br />
*5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample<br />
Running the gel<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*8 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 15 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*Very soft marks were observable for the gene ruler and the L. Plantarum sample<br />
<br />
<br />
----<br />
'''Experiment 26: Preparation of dishes with antibiotics (August 10th, 2013)'''<br />
<br />
*The LB agar was heated in the electrical stove until it was completely liquefied <br />
*The next plates were prepared:<br />
**4 control (agar only)<br />
**4 with kanamycin<br />
**2 with kanamycin and L-arabinose<br />
**2 with ampicillin<br />
*Plate with kanamycin<br />
**80 µl of kanamycin and 80 ml of LB agar were poured into a beaker (for 2 dishes)<br />
**They were mixed and poured into 4 dishes<br />
*Plate with kanamycin and L-arabinose<br />
**40 µl of kanamycin, 1200 µl of L-arabinose and 40 ml of LB agar were mixed in a beaker<br />
**The solution was poured into 2 petri dishes<br />
*Plate with ampicillin<br />
**40 µl of ampicillin and 40 ml of agar were mixed in a beaker<br />
**The solution was poured into 2 dishes<br />
*The mediums were prepared again to duplicate the amount of dishes<br />
*The plates solidified, properly labeled and refrigerated<br />
<br />
<br />
----<br />
'''Experiment 27: Transformation of E. Coli with linearized plasmids (psB4k5, psB1A7, psB2k3) (August 10th, 2013)'''<br />
<br />
*The competent cells were taken out of deep freeze and were left to defrost in ice<br />
*The competent cells were mixed softly and 50 µl of competent cells were placed in a cold eppendorf tube <br />
*2 µl of linearized plasmid were added and pipetted until it was homogeneous <br />
*It was sealed and left incubating in ice for 30 minutes<br />
*The tube was put through heat shock in water at 42⁰c for 60 seconds<br />
*The tube was placed in ice for 5 minutes<br />
*200 µl of S.O.C. were added<br />
*The tube was left incubating for 2 hours at 37⁰c and 150 rpm<br />
*(This was done with each plasmid)<br />
*Each plasmid was striated in a plate<br />
*They were sealed and left incubating at 37⁰c<br />
Results:<br />
*After an incubating period of 60 hours the plates with transformed E. Coli, plates were observed. Both plates presented bacterial growth. Therefore, the transformation was successful. Likewise, it was demonstrated that the previously prepared competent cells are effective. However, one dish presented a fungus, which was due to antibiotic which wasn’t filtered. Besides, the plates are not expressing GFP nor RFP<br />
<br />
<br />
----<br />
'''Experiment 28: Preparation of LB broth (August 12th, 2013)'''<br />
<br />
*6.0034 g of LB broth were weighted and diluted in 250 ml of distilled water. Afterwards, another 50 ml were added<br />
*A cotton and Kraft paper cap was used to cover the flask, and it was autoclaved for 15 minutes at 121⁰c<br />
<br />
<br />
----<br />
'''Experiment 29: Preparation of antibiotics (August 13th, 2013)'''<br />
<br />
*1.5 g of ampicillin were diluted in 15 ml of sterile water<br />
*0.750 g of kanamycin were weighted and diluted in 15 ml of sterile water<br />
*A syringe was used to its maximum capacity to hold the ampicillin stock<br />
*A Ministart hydrophilic filter 0.20µm was attached to the syringe<br />
*The content of the syringe was filtered into a falcon tube <br />
*The same procedure was used for the kanamycin stock<br />
*The stocks were sealed and stored in the refrigerator<br />
<br />
<br />
----<br />
'''Experiment 30: Purification of L. Plantarum plasmids (August 15th, 2013)'''<br />
<br />
*A sample from an isolated colony was used<br />
*1.5 ml of the sample was poured into an eppendorf tube<br />
*The tube was centrifuged for 4 minutes at 12000 rpm and the pellet was discarded<br />
*Another 1.5 ml of the samples were added to the tube<br />
*The tube was centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*250 µl of resuspension buffer (R3) were added and the sample was pipetted until the mixture was homogenous <br />
*The tube was incubated for 15 minutes<br />
*250 µl of Lysis buffer (L7) was added to the tube and mixed gently<br />
*It was left incubating for 5 minutes<br />
*350 µl of precipitation buffer were added and it was vigorously shaken<br />
*The tube was centrifuged at 12000 rpm for 10 minutes<br />
*The supernatant was transferred to a spin column in a 2 ml washtube <br />
*The tube was centrifuged for 1 minute at 12000 rpm<br />
*The liquid that passed through was discarded and the column was placed again in the washtube<br />
*500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute<br />
*The tube was centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded<br />
*700 µl of Wash buffer (W9) were added<br />
*The column was centrifuged at 12000 rpm for 1 minute<br />
*The supernatant was discarded and the column was transferred to an eppendorf tube of 1.5 ml <br />
*70 µl of TE buffer were added to the tube (previously heated at 65⁰c for 3 minutes)<br />
*The tube was incubated for 1 minute at room temperature<br />
*The tube was centrifuged at 12000 rpm for 2 minutes<br />
*The columns were discarded and the tubes were sealed and refrigerated at 4⁰c<br />
<br />
<br />
----<br />
'''Experiment 31: Gel for electrophoresis of L. Plantarum (purified plasmids) (August 15th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution <br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
*The agarose with the gel was heated in the microwave for 45 seconds<br />
Preparation of the sample to be placed in the gel<br />
*10 µl of each sample of purified plasmids were placed in eppendorf tubes of 1.5 ml<br />
*5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample<br />
Running the gel<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*15 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 20 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*There was no discernible mark other than the gene ruler <br />
<br />
<br />
----<br />
'''Experiment 32: Purification of L. Plantarum plasmids (August 15th, 2013)'''<br />
<br />
*A sample from an isolated colony was used<br />
*1.5 ml of the sample was poured into an eppendorf tube, a duplicate was made<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the pellet was discarded<br />
*Another 1.5 ml of the samples were added to the tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixture was homogenous <br />
*The tubes were incubated for 15 minutes<br />
*250 µl of Lysis buffer (L7) were added to the tubes and mixed gently<br />
*They were left incubating for 5 minutes<br />
*350 µl of precipitation buffer were added and it was vigorously shaken<br />
*The tubes were centrifuged at 12000 rpm for 10 minutes<br />
*The supernatant was transferred to a spin column in a 2 ml washtube <br />
*The tubes were centrifuged for 1 minute at 12000 rpm<br />
*The liquid that passed through was discarded and the column was placed again in the washtube<br />
*500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute<br />
*The tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded<br />
*700 µl of Wash buffer (W9) were added<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The supernatant was discarded and the columns were transferred to an eppendorf tube of 1.5 ml <br />
*70 µl of TE buffer were added to the tube (previously heated at 65⁰c for 3 minutes)<br />
*The tube was incubated for 11 minute at room temperature (because the centrifuge was occupied)<br />
*The tube was centrifuged at 12000 rpm for 2 minutes<br />
*The columns were discarded and the tubes were sealed and refrigerated at 4⁰c<br />
<br />
<br />
----<br />
'''Experiment 33: Purification of transformed E. Coli plasmids (psB3k5, psB4k5, psB1A7, psB2k3) (August 17th, 2013)'''<br />
<br />
*4 samples E. coli from an isolated colony were used<br />
*1.5 ml of each sample were poured into eppendorf tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the pellet was discarded<br />
*Another 1.5 ml of the samples were added to the tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixtures were homogenous <br />
*The tubes were incubated for 15 minutes<br />
*250 µl of Lysis buffer (L7) were added to each tube and mixed gently<br />
*They were left incubating for 5 minutes<br />
*350 µl of precipitation buffer were added and it was vigorously shaken<br />
*The tubes were centrifuged at 12000 rpm for 10 minutes<br />
*The supernatant was transferred to a spin column in a 2 ml washtube <br />
*The tubes were centrifuged for 1 minute at 12000 rpm<br />
*The liquid that passed through was discarded and the column was placed again in the washtube<br />
*500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute<br />
*Both tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded<br />
*700 µl of Wash buffer (W9) were added<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The supernatant was discarded and the columns were transferred to eppendorf tubes of 1.5 ml <br />
*70 µl of TE buffer were added to each tube (previously heated at 65⁰c for 3 minutes)<br />
*The tubes were incubated for 1 minute at room temperature<br />
*Both tubes were centrifuged at 12000 rpm for 2 minutes<br />
*The columns were discarded and the tubes were sealed and refrigerated at 4⁰c<br />
<br />
<br />
----<br />
'''Experiment 34: Gel for electrophoresis of L. Plantarum plasmids and plasmids (psB3k5, psB4k5, psB1A7, psB2k3) (August 17th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution <br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
*The agarose with the gel was heated in the microwave for 45 seconds<br />
Preparation of the sample to be placed in the gel<br />
*10 µl of each sample of purified plasmids were placed in eppendorf tubes of 1.5 ml<br />
*5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample<br />
Running the gel<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*15 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 20 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*There are marks in the gel; however most of the samples are blurred. The best defined marks are in track 7, at kbs. These marks are due to a faulty purification<br />
<br />
<br />
----<br />
'''Experiment 35: Purification of L. Plantarum plasmids and transformed E. Coli plasmids (psB3k5, psB4k5, psB1A7, psB2k3, psB1A7) (August 18th, 2013)'''<br />
<br />
*A sample of broth with L. Plantarum and LB with E. Coli was used for each corresponding plasmid<br />
*1.5 ml of each sample were poured into eppendorf tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the pellet was discarded<br />
*Another 1.5 ml of the samples were added to the tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixtures were homogenous <br />
*The tubes were incubated for 15 minutes<br />
*250 µl of Lysis buffer (L7) were added to each tube and mixed gently<br />
*They were left incubating for 5 minutes<br />
*350 µl of precipitation buffer were added and it was vigorously shaken<br />
*The tubes were centrifuged at 12000 rpm for 10 minutes<br />
*There was debris floating, so the supernatant was transferred to another tube and it was centrifuged again at 12000 rpm for 10 minutes<br />
*The supernatant was transferred to a spin column in a 2 ml washtube <br />
*The tubes were centrifuged for 1 minute at 12000 rpm<br />
*The liquid that passed through was discarded and the column was placed again in the washtube<br />
*500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute<br />
*Both tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded<br />
*700 µl of Wash buffer (W9) were added<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The supernatant was discarded and the columns were transferred to eppendorf tubes of 1.5 ml <br />
*70 µl of TE buffer were added to each tube (previously heated at 65⁰c for 3 minutes)<br />
*The tubes were incubated for 1 minute at room temperature<br />
*Both tubes were centrifuged at 12000 rpm for 2 minutes<br />
*The columns were discarded and the tubes were sealed and refrigerated at 4⁰c<br />
<br />
<br />
----<br />
'''Experiment 36: Gel for electrophoresis of purified plasmids (August 18th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution <br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
*The agarose with the gel was heated in the microwave for 45 seconds<br />
Preparation of the sample to be placed in the gel<br />
*10 µl of each sample of purified plasmids were placed in eppendorf tubes of 1.5 ml<br />
*5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample<br />
Running the gel (x2, because 2 gels were made)<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*15 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 20 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*Both gels presented intense marks in the tracks correspondent to the purified plasmids of E. Coli in the line of 20 kbs. The gels of L. Plantarum showed no marks<br />
<br />
<br />
----<br />
'''Experiment 37: Preparation of MRS broth, preparation of glycerol stocks and reactivation of glycerol stocks (August 19th, 2013)'''<br />
<br />
*10.2 g of MRS broth were weighted<br />
*The MRS broth was diluted in 200 ml of distilled water<br />
*The mediums were autoclaved for 15 min at 121⁰c<br />
*15 ml of MRS broth and 15 ml of glycerol (100%) were mixed in a 45 ml falcon tube, in the laminar flow cabinet<br />
*An aliquot of L. Plantarum was added to the solution and it was sealed with parafilm<br />
*1 glycerol stock was reactivated with MRS broth <br />
*A 1.5 ml stock was used with 15 ml of MRS broth, mixed in a falcon tube<br />
*Both tubes were left incubating at 37⁰c and 160 rpm<br />
<br />
<br />
----<br />
'''Experiment 38: Reactivation of the new L. Plantarum strain with glycerol stock (August 19th, 2013)'''<br />
<br />
*15 ml of MRS broth were mixed with 15 ml of glycerol (100%) in a 45 ml falcon tube<br />
*An aliquot of the new L. Plantarum was added and it was sealed with parafilm<br />
<br />
<br />
----<br />
'''E. Coli transformation with promoter 1,2 and rbs '''<br />
<br />
*Take the competent cells out of deep freeze and let them defrost in ice<br />
*Gently mix and put 50 µl in a cold 1.5 ml eppendorf tube<br />
*Add 10 µl of promoter 1, 2 and rbs <br />
* Gently mix them and leave them incubating in ice for 30 minutes<br />
*Cause the tube to go through heat shock, placing it in water at 42⁰c for 45 seconds<br />
*Cool the tube in ice for 5 minutes<br />
*Add 900 µl of S.O.C. to the tube<br />
*Gently mix it and leave it incubating for 1 hour at 37⁰c and 150 rpm <br />
*Striate a control dish and a dish with antibiotic <br />
*Seal both dishes and leave them incubating facing down at 37⁰c<br />
<br />
----<br />
Preparation of dishes with antibiotics (August 10th, 2013)<br />
The LB agar was heated in the electrical stove until it was completely liquefied<br />
The next plates were prepared:<br />
4 control (agar only)<br />
4 with kanamycin<br />
2 with kanamycin and L-arabinose<br />
2 with ampicillin<br />
Plate with kanamycin<br />
80 µl of kanamycin and 80 ml of LB agar were poured into a beaker (for 2 dishes)<br />
They were mixed and poured into 4 dishes<br />
Plate with kanamycin and L-arabinose<br />
40 µl of kanamycin, 1200 µl of L-arabinose and 40 ml of LB agar were mixed in a beaker<br />
The solution was poured into 2 petri dishes<br />
Plate with ampicillin<br />
40 µl of ampicillin and 40 ml of agar were mixed in a beaker<br />
The solution was poured into 2 dishes<br />
The mediums were prepared again to duplicate the amount of dishes<br />
The plates solidified, properly labeled and refrigerated</div>Gabyqzhttp://2013.igem.org/Team:BIOINT_Mexico/Lab_workTeam:BIOINT Mexico/Lab work2013-09-28T04:04:15Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Lab work</h1> </html><br />
----<br />
----<br />
<br />
'''Experiment 1: Preparation of growth mediums (MRS and LB) (June 5th, 2013)'''<br />
<br />
10 Agar LB plates<br />
<br />
15 Agar MRS plates<br />
<br />
<br />
Notes:<br />
*(1) 18.6g of Agar MRS were weighted to prepare 300ml of medium<br />
*(2) 7g of Agar LB were weighted to prepare 200ml of medium<br />
*(1) 5g of A-MRS were diluted in 80ml of distilled water<br />
*Another 5g and 170ml of distilled water were added<br />
*Small lumps were formed in the bottom of the Erlenmeyer flask<br />
*(2) 7g of Agar LB were diluted in 100ml of distilled water<br />
*Further 100ml of distilled water were added<br />
*It wasn’t possible to use a control of the sterilization because there was no autoclave tape<br />
*The autoclave began heating up<br />
*The autoclave valve was lowered<br />
*When the temperature of the autoclave reached 120⁰c, the power was cut to the half<br />
*The vapor cycle ended and the temperature decreased<br />
*The temperature reached 105⁰c and the valve was opened. The mediums were left inside the autoclave until de laminar flow cabinet was unoccupied<br />
*The medium in petri dishes were stored in the refrigerator <br />
*The growth mediums in storage were revised, they do not show signs of contamination <br />
17 MRS Agar<br />
<br />
9 LB Agar<br />
<br />
<br />
----<br />
'''Experiment 2: Preparation of MRS broth and glycerol stocks (July 8th, 2013)'''<br />
<br />
*0.7679g of powder for MRS broth were weighted<br />
*15ml of distilled water were measured in a 100ml measuring cylinder <br />
*Besides, 200ml were prepared for future use<br />
*10.009g of powder for MRS broth were weighted<br />
*It was diluted in 100ml of distilled water<br />
*Another 100ml of distilled water were added<br />
*0.7679g of powder for MRS broth were diluted in 15ml of distilled water<br />
*Both flasks were marked with autoclave tape and they were sterilized for 15 minutes at 121⁰c<br />
*They cooled down and the autoclave tape confirmed the sterilization<br />
*The 200ml broth was stored properly labeled <br />
*The 15ml of MRS broth and the 15ml of glycerol were mixed to 100% in the laminar flow cabinet. They were gently shaken until a homogenous mixture was obtained in a falcon tube of 45ml<br />
*An aliquot of L. Plantarum was added to the broth and glycerol solution and it was sealed with parafilm<br />
*It was left incubating at 37⁰c and 200rpm<br />
*After 24 hours the sample was taken out of the incubator to striate it<br />
*2500 (x2)ml were placed in 22 eppendorf tubes using a micropipette of 200µl to cultivate and recompile the mixture of L. Plantarum contained in the falcon tube <br />
*1 eppendorf tube was centrifuged at 13.4 rpm for 3 minutes <br />
*After the centrifugation there was a small pellet left, from which the supernatant was discarded and a solution of MRS broth (500ml) was added <br />
*Once the mixture was made, two petri dishes with MRS Agar were striated in the laminar flow cabinet. A third dish was striated in case of an emergency<br />
*The petri dishes were placed facing down in the incubator at 37⁰c and 200 rpm<br />
*The remaining samples (21 falcon tubes) were stored in deep freezing <br />
*E. Coli K12 was striated in two petri dishes with LB Agar <br />
*They were placed in the incubator at 37⁰c.<br />
<br />
<br />
----<br />
'''Experiment 3: Plasmid purification (July 16th, 2013)'''<br />
<br />
*An eppendorf tube of 1.5 ml with L. Plantarum culture was centrifuged at 12000 rpm for 1 minute two times<br />
*The supernatant was discarded and the tube’s content was resuspended with more L. Plantarum culture in MRS broth<br />
*The eppendorf tube was centrifuged for 15min at 6000 rpm<br />
*The supernatant was discarded <br />
*The pellet was resuspended in 250 µl resuspension buffer<br />
*250 µl of Lysis Buffer L7<br />
*The tube was gently mixed inverting it 5 times carefully<br />
*350 µl of precipitation Buffer N4 were added and mixed softly inverting the tube<br />
*It was centrifuged at 12000 rpm for 10 minutes<br />
*The supernatant was transferred to a spin column inside a washtube <br />
*It was centrifuged at 12000 rpm for a minute<br />
*The supernatant was discarded and 500 µl of Wash Buffer with ethanol (w10) were added to the column<br />
*It was incubated for one minute at room temperature<br />
*The column was centrifuged at 12000 rpm for 1 minute<br />
*The liquid from the washtube was discarded and the column was placed inside the tube<br />
*700 µl of Wash Buffer W9 with ethanol were added to the column<br />
*The column with the washtube was centrifuged at 12000 rpm for 1 minute<br />
*The liquid from the washtube was discarded<br />
*The column in the washtube was centrifuged at 12000 rpm for 1 minute<br />
*The liquid from the washtube was discarded<br />
*The column was placed inside an eppendorf tube of 1.5ml<br />
*75 µl of preheated TE Buffer were added at the center of the column<br />
*The TE Buffer was previously warmed in water bath at 65⁰c-70⁰c for 3 minutes<br />
*The column was incubated for 1 minute at room temperature<br />
*The column was centrifuged at 12000 rpm for 2 minutes<br />
*The eppendorf tube contains the purified plasmid<br />
<br />
<br />
----<br />
'''Experiment 4: Preparation of mediums for lactobacillus and E. Coli (July 17th, 2013)'''<br />
<br />
*15.3 g of MRS were weighted for 300 ml, the 2% (0.306 g.) of the MRS was used to do agar-agar<br />
*The autoclave started to heat <br />
*8.75 g of LB agar were weighted to dilute in 500 ml of distilled water<br />
*0.5005 g of LB broth were weighted to be diluted in 250 ml of distilled water<br />
*The 3 mediums were introduced in the autoclave<br />
*After 54 minutes the sterilization was complete <br />
*0.750 g of Kanamycin sulfate were measured and they were diluted in 15 ml of distilled water<br />
*1.5 g of Ampicillium sodium were weighted and diluted in 15 ml<br />
*The mediums were collocated in the laminar flow cabinet to pour into the petri dishes and striate <br />
*The mediums never solidified <br />
*2% more of agar was added in the LB broth and the mediums were placed in the autoclave <br />
*The mediums were collocated in the laminar flow cabinet to be poured into the petri dishes and striate<br />
*But the mediums didn’t solidified<br />
<br />
<br />
----<br />
'''Experiment 5: Preparation of MRS agar mediums (July 19th, 2013)'''<br />
<br />
*The mediums didn’t solidify, because the agar added wasn’t enough<br />
*10.2 g of MRS broth were measured and 2 g of agar-agar were added<br />
*The pH of the mediums was 6.6 <br />
*A stock of HCl at 10% was added until the pH reached 6.4 <br />
*The mediums were sterilized with the autoclave for 15 minutes at 121°C <br />
*40 ml of agar were mixed with 40 µl of stock of ampicillin and they were poured into 2 petri dishes<br />
*40 ml of MRS agar were mixed with 40 µl of kanamycin stock and they were poured into 2 petri dishes <br />
*6 petri dishes were made with only MRS agar <br />
*2 control petri dishes with MRS agar were striated with L. Plantarum with 1.5 ml of glycerol stock <br />
*The petri dishes were incubated at 37°C for 2 hours<br />
<br />
<br />
----<br />
'''Experiment 6: Growth of E. Coli (July 21st, 2013)'''<br />
<br />
*No growth was discernible in the petri dishes with E. Coli, which were left incubating. This attributed to the use of an E. Coli sample, which was in refrigeration since 2011<br />
*The LB Agar medium was heated in an electric stove for approximately 10 minutes<br />
*A new sample of lyophilized E. Coli sample was opened and a representative sample was extracted to be rehydrated with LB broth<br />
*Liquid LB agar was poured into 2 petri dishes and they were left to solidify<br />
*Both dishes were striated with the rehydrated E. Coli<br />
*Both dishes were left incubating, after being sealed, during 24 hours at 37⁰c<br />
<br />
<br />
----<br />
'''Experiment 7: Glycerol stocks reactivation (July 22nd, 2013)'''<br />
<br />
*No discernible growth was observed in the L. Plantarum plates after a 72 hours incubation period. After consulting an external source, it was determined that this was due to glycerol stocks used, which weren’t reactivated<br />
*Two stocks of glycerol were reactivated with MRS broth<br />
*10 ml of MRS broth were added to a 1 ml stock in a falcon tube<br />
*The second stock was poured into another falcon tube, the result was 1.5 ml of stock with 15 ml of MRS broth<br />
*Both falcon tubes were sealed and left incubating at 37⁰c<br />
<br />
<br />
----<br />
'''Experiment 8: Overnight growth of E. Coli in LB broth (July 23rd, 2013)''' <br />
<br />
*5 ml of LB broth were poured into 2 falcon tubes each <br />
*An E. Coli colony was added to each tube with broth <br />
*Both tubes were sealed and left incubating at 37⁰c<br />
<br />
<br />
----<br />
'''Experiment 9: Preparation of mediums for lactobacillus and E. Coli (July 24th, 2013)'''<br />
<br />
*15.3 g of MRS and 6 g of bacto agar were weighted to be diluted in 300 ml<br />
*17.5 g of LB agar were weighted to prepare 500 ml<br />
*The MRS and LB agar were autoclaved <br />
*1 ml of E. Coli was inoculated in 100 ml of LB agar in a flask of 250 ml<br />
*5 g of LB broth were weighted<br />
*It was autoclaved for 15 minutes at 121⁰c<br />
<br />
<br />
----<br />
'''Experiment 10: Preparation of TE buffer (July 26th, 2013)'''<br />
<br />
*6.05 g of TRIS were weighted and diluted in 60 ml of distilled water<br />
*9.3041 g of EDTA were weighted and diluted in 60 ml of distilled water<br />
*The TRIS’ pH was 10.29, therefore HCl was added until the pH reached 8.3<br />
*The EDTA’s pH was 3.25, therefore NaOH crystals were added until the pH reached 7.79<br />
*They were sealed and autoclaved for 15 minutes at 121⁰c<br />
<br />
<br />
----<br />
'''Experiment 11: Preparation of competent E. Coli cells (July 28th, 2013)'''<br />
<br />
*A falcon tube was prepared with 50 ml of CaCl2 0.1M<br />
*Another falcon tube was prepared with CaCl2 0.1M/ 15% glycerol<br />
*4 eppendorf tubes of 1.5 ml were filled <br />
*The 4 tubes were left in ice for 10 minutes<br />
*The 4 tubes were centrifuged for 3 minutes at 6000 rpm and the supernatant was discarded<br />
*1.5 ml of the aforementioned culture were added to each tube<br />
*The tubes were centrifuged for 3 minutes at 6000 rpm <br />
*The supernatant was discarded<br />
*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M for each tube<br />
*The 4 tubes were incubated in ice for 20 minutes<br />
*The tubes were centrifuged again for 3 minutes at 6000 rpm<br />
*The supernatant was discarded<br />
*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each<br />
*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c<br />
<br />
<br />
----<br />
'''Experiment 12: L. Plantarum plasmid purification (July 28th, 2013)'''<br />
<br />
*1.5 ml of L. Plantarum culture in MRS broth were poured into 2 eppendorf tubes, 1.5 ml each<br />
*Both tubes were centrifuged for 4 minutes at 12000 rpm<br />
*The supernatant was discarded and another 1.5 ml of culture were added <br />
*Both tubes were centrifuged for 4 minutes at 12000 rpm<br />
*The supernatant was discarded <br />
*250 µl of resuspension buffer (R3) were added and the pellets were resuspended until the mixture was homogenous <br />
*The tubes were incubated at room temperature for 15 minutes<br />
*250 µl of Lysis buffer (L7) were added to each tube. They were gently mixed<br />
*The tubes were incubated at room temperature for 5 minutes<br />
*350 µl of precipitation buffer (N4) were added <br />
*Both tubes were vigorously mixed until a homogenous mixture was obtained <br />
*The tubes were centrifuged at 12000 rpm for 10 minutes<br />
*The supernatant was poured into a spin column in a wash tube of 2 ml <br />
*The columns were centrifuged at 12000 rpm for 1 minute <br />
*The supernatant was discarded and the column was placed in the new washtube<br />
*500 µl of wash buffer (W10) with ethanol were added to the column <br />
*The columns were incubated for 1 minute at room temperature<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The liquid that went through the columns was discarded<br />
*700 µl of wash buffer (W9) were added<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The liquid that went through the columns was discarded<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The liquid that went through the columns and the washtube was discarded<br />
*The columns were placed in eppendorf tubes of 1.5 ml<br />
*70 µl of TE buffer (previously heated at 65⁰c for 3 minutes) were added in the center of each column<br />
*The columns were incubated at room temperature for 1 minute <br />
*The columns were centrifuged at 12000 rpm for 2 minutes<br />
*The eppendorf tubes contain the purified plasmids<br />
*The columns were removed, the eppendorf tubes were sealed and they were refrigerated at 4⁰c until the next day<br />
<br />
<br />
----<br />
'''Experiment 13: Electrophoresis gel for L. Plantarum (July 29th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution <br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
<br />
Preparation of the sample to be placed in the gel<br />
*10 µl of each sample of purified plasmid were placed in eppendorf tubes of 1.5 ml<br />
*5 µl of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample<br />
<br />
Running the gel<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*15 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 15 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*No sample presented marks in the gel, only the gene ruler<br />
<br />
<br />
----<br />
'''Experiment 14: Overnight culture of L. Plnatarum and agarose preparation (July 29th, 2013)'''<br />
<br />
*2 colonies with one day of incubation were inoculated in MRS broth out of a 10 ml sample<br />
*They were placed in falcon tubes and left incubating at 37.2⁰c<br />
*35 ml of a TAE 1x solution were used to prepare agarose gel for electrophoresis. 0.35 g of agarose were added to the solution to solidify it<br />
*The liquid gel was poured into a casting tray and the well combs were placed<br />
<br />
<br />
----<br />
'''Experiment 15: Rehydration of plasmids in kit plates (July 30th, 2013)'''<br />
<br />
*The plate kits were numbered as indicated in the iGEM’s parts registry and the plasmids to be used were localized<br />
*The protective layer was pierced with a micro-pipet<br />
*Each plasmid was rehydrated with 10 µl of distilled water<br />
*The liquid was pipetted until it was homogeneous <br />
*It was left resting for 5 minutes to ensure its rehydration<br />
<br />
<br />
----<br />
'''Experiment 16: Electrophoresis gel for kit plates plasmids (July 30th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to form a 400 ml solution<br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
*The agarose with TAE 1x was heated in the microwave for 4 seconds<br />
Preparation of the sample to be run in gel<br />
*5 µl of each rehydrated plasmid were placed in eppendorf tubes <br />
*10 µl of 6x DNA Loading Dye were added and it was pipetted until the mixture was homogeneous<br />
Running the gel<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*15 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 15 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*Thin marks can be appreciated in samples psB4A5 and psB1A7<br />
<br />
<br />
----<br />
'''Experiment 17: E. Coli transformation with linearized plasmids (psB4k5, psB1k3, psB2k3) (July 31st, 2013)'''<br />
<br />
*The competent cells were taken out of deep freeze and were left to defrost in ice<br />
*They were mixed softly and 50 µl of competent cells were placed in a cold eppendorf tube <br />
*10 µl of linearized plasmid were added (psB4k5)<br />
*It was mixed gently and left incubating in ice for 30 minutes<br />
*The tube was put through heat shock in water at 42⁰c for 45 seconds<br />
*The tube was placed in ice for 2-5 minutes<br />
*900 µl of S.O.C. were added<br />
*The tube was left incubating for 1 hour at 37⁰c and 200 rpm<br />
*A dish with LB agar and kanamycin was striated with the transformed E. Coli<br />
*The dish was sealed and left incubating at 37⁰c<br />
*The same procedure was followed for plasmids psB1k3 and psB2k3<br />
<br />
<br />
----<br />
'''Experiment 18: Preparation of transformation buffer CaCl2 50mM (August 2nd, 2013)'''<br />
<br />
*1 ml of CaCl2 buffer 200mM were placed in a beaker<br />
*3 ml of sterile distilled water were added<br />
*With a syringe, it was filled to its maximum capacity with the buffer<br />
*A ministart 0.2µm filter was connected to the syringe<br />
*The content of the syringe was poured into a sterile falcon tube through the tube <br />
*The tube was left cooling in ice<br />
<br />
<br />
----<br />
'''Experiment 19: E. Coli transformation with pGLO (GFP) (August 3rd, 2013)'''<br />
<br />
*The competent cells were taken out of deep freeze and left defrosting in ice<br />
*They were gently mixed and 50 µl were placed in a cold 1.5 ml eppendorf tube<br />
*10 µl of pGLO were added<br />
*They were gently mixed and left incubating in ice for 30 minutes<br />
*The tube went through heat shock in water at 42⁰c for 45 seconds<br />
*The tube was left in ice for 5 minutes<br />
*900 µl of S.O.C. was added to the tube<br />
*It was gently mixed and left incubating for 1 hour at 37⁰c and 150 rpm <br />
*A control dish and a dish with antibiotic were striated <br />
*Both dishes were sealed and left incubating facing down at 37⁰c<br />
<br />
<br />
----<br />
'''Experiment 20: MRS and LB mediums preparation (August 5th, 2013)'''<br />
<br />
*15.3 g of MRS and 6 g of bacto agar were weighted to be diluted in 300 ml<br />
*17.5 g of LB agar were weighted to prepare 500 ml<br />
*The MRS and LB agar were autoclaved for 15 minutes at 121⁰c<br />
*They were left in refrigeration<br />
Results:<br />
*After an incubating period of 24 hours both, the control and the L-arabinose and ampicillin, plates were observed. Both plates presented bacterial growth. The bacteria in the L-arabinose plate were analyzed in the transilluminator and they glowed. Therefore, the transformation was successful and the gen was expressed correctly. Likewise, it was demonstrated that the previously prepared competent cells are effective. <br />
<br />
<br />
----<br />
'''Experiment 21: Preparation of plates with LB agar and antibiotic (August 6th, 2013)'''<br />
<br />
*The LB agar was heated in the electrical stove until it was completely liquefied <br />
*The next plates were prepared:<br />
**1 control (only agar)<br />
**2 with kanamycin<br />
**1 with kanamycin and L-arabinose<br />
**1 with ampicillin<br />
*Plate with kanamycin<br />
**40 µl of kanamycin and 40 ml of LB agar were poured into a beaker (for 2 dishes)<br />
**They were mixed and poured into 2 dishes<br />
*Plate with kanamycin and L-arabinose<br />
**20 µl of kanamycin, 600 µl of L-arabinose and 20 ml of LB agar were mixed in a beaker<br />
**The solution was poured into a petri dish<br />
*Plate with ampicillin<br />
**20 µl of ampicillin and 20 ml of agar were mixed in a beaker<br />
**The solution was poured into a dish<br />
*The mediums were prepared again to duplicate the amount of dishes<br />
*The plates solidified and they were properly labeled<br />
*A total of 9 dishes were prepared<br />
**1 with LB agar<br />
**4 with kanamycin<br />
**2 with kanamycin and L-arabinose<br />
**2 with ampicillin<br />
<br />
<br />
----<br />
'''Experiment 22: E. Coli transformation with linearized plasmids (psB4k5, psB1A7, psB2k3) (August 7th, 2013)'''<br />
<br />
*The DNA of each plasmid was rehydrated with 10 µl of sterile distilled water<br />
*Each well of the kit plates was pierced with a micro pipet<br />
*Each rehydrated DNA was stored in eppendorf tubes<br />
*The competent cells were taken out of deep freeze and were left to defrost in ice<br />
*The competent cells were mixed softly and 50 µl of competent cells were placed in a cold eppendorf tube <br />
*2 µl of linearized plasmid were added and pipetted until it was homogeneous <br />
*It was sealed and left incubating in ice for 30 minutes<br />
*The tube was put through heat shock in water at 42⁰c for 60 seconds<br />
*The tube was placed in ice for 5 minutes<br />
*200 µl of S.O.C. were added<br />
*The tube was left incubating for 2 hours at 37⁰c<br />
*(This was done with each plasmid)<br />
*The samples were striated with a Digralsky spreader in dishes with their correspondent antibiotic and/or inducer<br />
*For each plasmid it was inoculated:<br />
##In a dish, 20µl of a sample of transformed E. Coli (with antibiotics)<br />
##In a dish, 100 µl of the same transformed E. Coli (with antibiotics)<br />
##In a control dish, the rest of the transformed cells<br />
*they were sealed and left incubating at 37⁰c<br />
<br />
<br />
----<br />
'''Experiment 23: Preparation of competent cells with Lactobacillus Plantarum (August 8th, 2013)'''<br />
<br />
*2 eppendorf tubes of 1.5 ml were filled with L. Plantarum in MRS broth<br />
*The tubes were left in ice for 10 minutes<br />
*The tubes were centrifuged for 3 minutes at 6000 rpm and the supernatant was discarded<br />
*1.5 ml of the aforementioned culture were added to each tube<br />
*The tubes were centrifuged for 3 minutes at 6000 rpm <br />
*The supernatant was discarded<br />
*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M for each tube<br />
*The tubes were incubated in ice for 20 minutes<br />
*The tubes were centrifuged again for 3 minutes at 6000 rpm<br />
*The supernatant was discarded<br />
*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each<br />
*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c<br />
<br />
<br />
----<br />
'''Experiment 24: Purification of L. Plantarum plasmids (August 9th, 2013)'''<br />
<br />
*2 different samples of L. Plantarum were used, one from an isolated colony and another form a reactivated stock<br />
*1.5 ml of each sample were poured into eppendorf tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*Another 1.5 ml of the samples were added to the tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixtures were homogenous <br />
*The tubes were incubated for 15 minutes<br />
*250 µl of Lysis buffer (L7) were added to each tube and mixed gently<br />
*They were left incubating for 5 minutes<br />
*350 µl of precipitation buffer were added and it was vigorously shaken<br />
*Both tubes were centrifuged at 12000 rpm for 10 minutes<br />
*It was observed that before centrifuging the samples, the tube with the reactivated cells had a much larger volume than the other, which meant a mistake was probably made when pipetting <br />
*The supernatant was transferred to a spin column in a 2 ml washtube <br />
*The tubes were centrifuged for 1 minute at 12000 rpm<br />
*The liquid that passed through was discarded and the column was placed again in the washtube<br />
*500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute<br />
*Both tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded<br />
*700 µl of Wash buffer (W9) were added<br />
*Both columns were centrifuged at 12000 rpm for 1 minute<br />
*The supernatant was discarded and the columns were transferred to eppendorf tubes of 1.5 ml <br />
*70 µl of TE buffer were added to each tube (previously heated at 65⁰c for 3 minutes)<br />
*The tubes were incubated for 1 minute at room temperature<br />
*Both tubes were centrifuged at 12000 rpm for 2 minutes<br />
*The columns were discarded and the tubes were sealed and refrigerated at 4⁰c<br />
<br />
<br />
----<br />
'''Experiment 25: Gel for electrophoresis of plasmids (August 9th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution <br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
*The agarose with the gel was heated in the microwave for 45 seconds<br />
Preparation of the sample to be placed in the gel<br />
*10 µl of each sample of purified plasmid were placed in eppendorf tubes of 1.5 ml<br />
*5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample<br />
Running the gel<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*8 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 15 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*Very soft marks were observable for the gene ruler and the L. Plantarum sample<br />
<br />
<br />
----<br />
'''Experiment 26: Preparation of dishes with antibiotics (August 10th, 2013)'''<br />
<br />
*The LB agar was heated in the electrical stove until it was completely liquefied <br />
*The next plates were prepared:<br />
**4 control (agar only)<br />
**4 with kanamycin<br />
**2 with kanamycin and L-arabinose<br />
**2 with ampicillin<br />
*Plate with kanamycin<br />
**80 µl of kanamycin and 80 ml of LB agar were poured into a beaker (for 2 dishes)<br />
**They were mixed and poured into 4 dishes<br />
*Plate with kanamycin and L-arabinose<br />
**40 µl of kanamycin, 1200 µl of L-arabinose and 40 ml of LB agar were mixed in a beaker<br />
**The solution was poured into 2 petri dishes<br />
*Plate with ampicillin<br />
**40 µl of ampicillin and 40 ml of agar were mixed in a beaker<br />
**The solution was poured into 2 dishes<br />
*The mediums were prepared again to duplicate the amount of dishes<br />
*The plates solidified, properly labeled and refrigerated<br />
<br />
<br />
----<br />
'''Experiment 27: Transformation of E. Coli with linearized plasmids (psB4k5, psB1A7, psB2k3) (August 10th, 2013)'''<br />
<br />
*The competent cells were taken out of deep freeze and were left to defrost in ice<br />
*The competent cells were mixed softly and 50 µl of competent cells were placed in a cold eppendorf tube <br />
*2 µl of linearized plasmid were added and pipetted until it was homogeneous <br />
*It was sealed and left incubating in ice for 30 minutes<br />
*The tube was put through heat shock in water at 42⁰c for 60 seconds<br />
*The tube was placed in ice for 5 minutes<br />
*200 µl of S.O.C. were added<br />
*The tube was left incubating for 2 hours at 37⁰c and 150 rpm<br />
*(This was done with each plasmid)<br />
*Each plasmid was striated in a plate<br />
*They were sealed and left incubating at 37⁰c<br />
Results:<br />
*After an incubating period of 60 hours the plates with transformed E. Coli, plates were observed. Both plates presented bacterial growth. Therefore, the transformation was successful. Likewise, it was demonstrated that the previously prepared competent cells are effective. However, one dish presented a fungus, which was due to antibiotic which wasn’t filtered. Besides, the plates are not expressing GFP nor RFP<br />
<br />
<br />
----<br />
'''Experiment 28: Preparation of LB broth (August 12th, 2013)'''<br />
<br />
*6.0034 g of LB broth were weighted and diluted in 250 ml of distilled water. Afterwards, another 50 ml were added<br />
*A cotton and Kraft paper cap was used to cover the flask, and it was autoclaved for 15 minutes at 121⁰c<br />
<br />
<br />
----<br />
'''Experiment 29: Preparation of antibiotics (August 13th, 2013)'''<br />
<br />
*1.5 g of ampicillin were diluted in 15 ml of sterile water<br />
*0.750 g of kanamycin were weighted and diluted in 15 ml of sterile water<br />
*A syringe was used to its maximum capacity to hold the ampicillin stock<br />
*A Ministart hydrophilic filter 0.20µm was attached to the syringe<br />
*The content of the syringe was filtered into a falcon tube <br />
*The same procedure was used for the kanamycin stock<br />
*The stocks were sealed and stored in the refrigerator<br />
<br />
<br />
----<br />
'''Experiment 30: Purification of L. Plantarum plasmids (August 15th, 2013)'''<br />
<br />
*A sample from an isolated colony was used<br />
*1.5 ml of the sample was poured into an eppendorf tube<br />
*The tube was centrifuged for 4 minutes at 12000 rpm and the pellet was discarded<br />
*Another 1.5 ml of the samples were added to the tube<br />
*The tube was centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*250 µl of resuspension buffer (R3) were added and the sample was pipetted until the mixture was homogenous <br />
*The tube was incubated for 15 minutes<br />
*250 µl of Lysis buffer (L7) was added to the tube and mixed gently<br />
*It was left incubating for 5 minutes<br />
*350 µl of precipitation buffer were added and it was vigorously shaken<br />
*The tube was centrifuged at 12000 rpm for 10 minutes<br />
*The supernatant was transferred to a spin column in a 2 ml washtube <br />
*The tube was centrifuged for 1 minute at 12000 rpm<br />
*The liquid that passed through was discarded and the column was placed again in the washtube<br />
*500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute<br />
*The tube was centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded<br />
*700 µl of Wash buffer (W9) were added<br />
*The column was centrifuged at 12000 rpm for 1 minute<br />
*The supernatant was discarded and the column was transferred to an eppendorf tube of 1.5 ml <br />
*70 µl of TE buffer were added to the tube (previously heated at 65⁰c for 3 minutes)<br />
*The tube was incubated for 1 minute at room temperature<br />
*The tube was centrifuged at 12000 rpm for 2 minutes<br />
*The columns were discarded and the tubes were sealed and refrigerated at 4⁰c<br />
<br />
<br />
----<br />
'''Experiment 31: Gel for electrophoresis of L. Plantarum (purified plasmids) (August 15th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution <br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
*The agarose with the gel was heated in the microwave for 45 seconds<br />
Preparation of the sample to be placed in the gel<br />
*10 µl of each sample of purified plasmids were placed in eppendorf tubes of 1.5 ml<br />
*5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample<br />
Running the gel<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*15 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 20 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*There was no discernible mark other than the gene ruler <br />
<br />
<br />
----<br />
'''Experiment 32: Purification of L. Plantarum plasmids (August 15th, 2013)'''<br />
<br />
*A sample from an isolated colony was used<br />
*1.5 ml of the sample was poured into an eppendorf tube, a duplicate was made<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the pellet was discarded<br />
*Another 1.5 ml of the samples were added to the tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixture was homogenous <br />
*The tubes were incubated for 15 minutes<br />
*250 µl of Lysis buffer (L7) were added to the tubes and mixed gently<br />
*They were left incubating for 5 minutes<br />
*350 µl of precipitation buffer were added and it was vigorously shaken<br />
*The tubes were centrifuged at 12000 rpm for 10 minutes<br />
*The supernatant was transferred to a spin column in a 2 ml washtube <br />
*The tubes were centrifuged for 1 minute at 12000 rpm<br />
*The liquid that passed through was discarded and the column was placed again in the washtube<br />
*500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute<br />
*The tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded<br />
*700 µl of Wash buffer (W9) were added<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The supernatant was discarded and the columns were transferred to an eppendorf tube of 1.5 ml <br />
*70 µl of TE buffer were added to the tube (previously heated at 65⁰c for 3 minutes)<br />
*The tube was incubated for 11 minute at room temperature (because the centrifuge was occupied)<br />
*The tube was centrifuged at 12000 rpm for 2 minutes<br />
*The columns were discarded and the tubes were sealed and refrigerated at 4⁰c<br />
<br />
<br />
----<br />
'''Experiment 33: Purification of transformed E. Coli plasmids (psB3k5, psB4k5, psB1A7, psB2k3) (August 17th, 2013)'''<br />
<br />
*4 samples E. coli from an isolated colony were used<br />
*1.5 ml of each sample were poured into eppendorf tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the pellet was discarded<br />
*Another 1.5 ml of the samples were added to the tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixtures were homogenous <br />
*The tubes were incubated for 15 minutes<br />
*250 µl of Lysis buffer (L7) were added to each tube and mixed gently<br />
*They were left incubating for 5 minutes<br />
*350 µl of precipitation buffer were added and it was vigorously shaken<br />
*The tubes were centrifuged at 12000 rpm for 10 minutes<br />
*The supernatant was transferred to a spin column in a 2 ml washtube <br />
*The tubes were centrifuged for 1 minute at 12000 rpm<br />
*The liquid that passed through was discarded and the column was placed again in the washtube<br />
*500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute<br />
*Both tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded<br />
*700 µl of Wash buffer (W9) were added<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The supernatant was discarded and the columns were transferred to eppendorf tubes of 1.5 ml <br />
*70 µl of TE buffer were added to each tube (previously heated at 65⁰c for 3 minutes)<br />
*The tubes were incubated for 1 minute at room temperature<br />
*Both tubes were centrifuged at 12000 rpm for 2 minutes<br />
*The columns were discarded and the tubes were sealed and refrigerated at 4⁰c<br />
<br />
<br />
----<br />
'''Experiment 34: Gel for electrophoresis of L. Plantarum plasmids and plasmids (psB3k5, psB4k5, psB1A7, psB2k3) (August 17th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution <br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
*The agarose with the gel was heated in the microwave for 45 seconds<br />
Preparation of the sample to be placed in the gel<br />
*10 µl of each sample of purified plasmids were placed in eppendorf tubes of 1.5 ml<br />
*5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample<br />
Running the gel<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*15 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 20 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*There are marks in the gel; however most of the samples are blurred. The best defined marks are in track 7, at kbs. These marks are due to a faulty purification<br />
<br />
<br />
----<br />
'''Experiment 35: Purification of L. Plantarum plasmids and transformed E. Coli plasmids (psB3k5, psB4k5, psB1A7, psB2k3, psB1A7) (August 18th, 2013)'''<br />
<br />
*A sample of broth with L. Plantarum and LB with E. Coli was used for each corresponding plasmid<br />
*1.5 ml of each sample were poured into eppendorf tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the pellet was discarded<br />
*Another 1.5 ml of the samples were added to the tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixtures were homogenous <br />
*The tubes were incubated for 15 minutes<br />
*250 µl of Lysis buffer (L7) were added to each tube and mixed gently<br />
*They were left incubating for 5 minutes<br />
*350 µl of precipitation buffer were added and it was vigorously shaken<br />
*The tubes were centrifuged at 12000 rpm for 10 minutes<br />
*There was debris floating, so the supernatant was transferred to another tube and it was centrifuged again at 12000 rpm for 10 minutes<br />
*The supernatant was transferred to a spin column in a 2 ml washtube <br />
*The tubes were centrifuged for 1 minute at 12000 rpm<br />
*The liquid that passed through was discarded and the column was placed again in the washtube<br />
*500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute<br />
*Both tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded<br />
*700 µl of Wash buffer (W9) were added<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The supernatant was discarded and the columns were transferred to eppendorf tubes of 1.5 ml <br />
*70 µl of TE buffer were added to each tube (previously heated at 65⁰c for 3 minutes)<br />
*The tubes were incubated for 1 minute at room temperature<br />
*Both tubes were centrifuged at 12000 rpm for 2 minutes<br />
*The columns were discarded and the tubes were sealed and refrigerated at 4⁰c<br />
<br />
<br />
----<br />
'''Experiment 36: Gel for electrophoresis of purified plasmids (August 18th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution <br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
*The agarose with the gel was heated in the microwave for 45 seconds<br />
Preparation of the sample to be placed in the gel<br />
*10 µl of each sample of purified plasmids were placed in eppendorf tubes of 1.5 ml<br />
*5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample<br />
Running the gel (x2, because 2 gels were made)<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*15 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 20 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*Both gels presented intense marks in the tracks correspondent to the purified plasmids of E. Coli in the line of 20 kbs. The gels of L. Plantarum showed no marks<br />
<br />
<br />
----<br />
'''Experiment 37: Preparation of MRS broth, preparation of glycerol stocks and reactivation of glycerol stocks (August 19th, 2013)'''<br />
<br />
*10.2 g of MRS broth were weighted<br />
*The MRS broth was diluted in 200 ml of distilled water<br />
*The mediums were autoclaved for 15 min at 121⁰c<br />
*15 ml of MRS broth and 15 ml of glycerol (100%) were mixed in a 45 ml falcon tube, in the laminar flow cabinet<br />
*An aliquot of L. Plantarum was added to the solution and it was sealed with parafilm<br />
*1 glycerol stock was reactivated with MRS broth <br />
*A 1.5 ml stock was used with 15 ml of MRS broth, mixed in a falcon tube<br />
*Both tubes were left incubating at 37⁰c and 160 rpm<br />
<br />
<br />
----<br />
'''Experiment 38: Reactivation of the new L. Plantarum strain with glycerol stock (August 19th, 2013)'''<br />
<br />
*15 ml of MRS broth were mixed with 15 ml of glycerol (100%) in a 45 ml falcon tube<br />
*An aliquot of the new L. Plantarum was added and it was sealed with parafilm<br />
<br />
<br />
----<br />
'''E. Coli transformation with promoter 1,2 and rbs '''<br />
<br />
*Take the competent cells out of deep freeze and let them defrost in ice<br />
*Gently mix and put 50 µl in a cold 1.5 ml eppendorf tube<br />
*Add 10 µl of promoter 1, 2 and rbs <br />
* Gently mix them and leave them incubating in ice for 30 minutes<br />
*Cause the tube to go through heat shock, placing it in water at 42⁰c for 45 seconds<br />
*Cool the tube in ice for 5 minutes<br />
*Add 900 µl of S.O.C. to the tube<br />
*Gently mix it and leave it incubating for 1 hour at 37⁰c and 150 rpm <br />
*Striate a control dish and a dish with antibiotic <br />
*Seal both dishes and leave them incubating facing down at 37⁰c</div>Gabyqzhttp://2013.igem.org/File:GFP3.jpgFile:GFP3.jpg2013-09-28T04:04:12Z<p>Gabyqz: </p>
<hr />
<div></div>Gabyqzhttp://2013.igem.org/Team:BIOINT_Mexico/Lab_workTeam:BIOINT Mexico/Lab work2013-09-28T04:03:04Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Lab work</h1> </html><br />
----<br />
----<br />
<br />
'''Experiment 1: Preparation of growth mediums (MRS and LB) (June 5th, 2013)'''<br />
<br />
10 Agar LB plates<br />
<br />
15 Agar MRS plates<br />
<br />
<br />
Notes:<br />
*(1) 18.6g of Agar MRS were weighted to prepare 300ml of medium<br />
*(2) 7g of Agar LB were weighted to prepare 200ml of medium<br />
*(1) 5g of A-MRS were diluted in 80ml of distilled water<br />
*Another 5g and 170ml of distilled water were added<br />
*Small lumps were formed in the bottom of the Erlenmeyer flask<br />
*(2) 7g of Agar LB were diluted in 100ml of distilled water<br />
*Further 100ml of distilled water were added<br />
*It wasn’t possible to use a control of the sterilization because there was no autoclave tape<br />
*The autoclave began heating up<br />
*The autoclave valve was lowered<br />
*When the temperature of the autoclave reached 120⁰c, the power was cut to the half<br />
*The vapor cycle ended and the temperature decreased<br />
*The temperature reached 105⁰c and the valve was opened. The mediums were left inside the autoclave until de laminar flow cabinet was unoccupied<br />
*The medium in petri dishes were stored in the refrigerator <br />
*The growth mediums in storage were revised, they do not show signs of contamination <br />
17 MRS Agar<br />
<br />
9 LB Agar<br />
<br />
<br />
----<br />
'''Experiment 2: Preparation of MRS broth and glycerol stocks (July 8th, 2013)'''<br />
<br />
*0.7679g of powder for MRS broth were weighted<br />
*15ml of distilled water were measured in a 100ml measuring cylinder <br />
*Besides, 200ml were prepared for future use<br />
*10.009g of powder for MRS broth were weighted<br />
*It was diluted in 100ml of distilled water<br />
*Another 100ml of distilled water were added<br />
*0.7679g of powder for MRS broth were diluted in 15ml of distilled water<br />
*Both flasks were marked with autoclave tape and they were sterilized for 15 minutes at 121⁰c<br />
*They cooled down and the autoclave tape confirmed the sterilization<br />
*The 200ml broth was stored properly labeled <br />
*The 15ml of MRS broth and the 15ml of glycerol were mixed to 100% in the laminar flow cabinet. They were gently shaken until a homogenous mixture was obtained in a falcon tube of 45ml<br />
*An aliquot of L. Plantarum was added to the broth and glycerol solution and it was sealed with parafilm<br />
*It was left incubating at 37⁰c and 200rpm<br />
*After 24 hours the sample was taken out of the incubator to striate it<br />
*2500 (x2)ml were placed in 22 eppendorf tubes using a micropipette of 200µl to cultivate and recompile the mixture of L. Plantarum contained in the falcon tube <br />
*1 eppendorf tube was centrifuged at 13.4 rpm for 3 minutes <br />
*After the centrifugation there was a small pellet left, from which the supernatant was discarded and a solution of MRS broth (500ml) was added <br />
*Once the mixture was made, two petri dishes with MRS Agar were striated in the laminar flow cabinet. A third dish was striated in case of an emergency<br />
*The petri dishes were placed facing down in the incubator at 37⁰c and 200 rpm<br />
*The remaining samples (21 falcon tubes) were stored in deep freezing <br />
*E. Coli K12 was striated in two petri dishes with LB Agar <br />
*They were placed in the incubator at 37⁰c.<br />
<br />
<br />
----<br />
'''Experiment 3: Plasmid purification (July 16th, 2013)'''<br />
<br />
*An eppendorf tube of 1.5 ml with L. Plantarum culture was centrifuged at 12000 rpm for 1 minute two times<br />
*The supernatant was discarded and the tube’s content was resuspended with more L. Plantarum culture in MRS broth<br />
*The eppendorf tube was centrifuged for 15min at 6000 rpm<br />
*The supernatant was discarded <br />
*The pellet was resuspended in 250 µl resuspension buffer<br />
*250 µl of Lysis Buffer L7<br />
*The tube was gently mixed inverting it 5 times carefully<br />
*350 µl of precipitation Buffer N4 were added and mixed softly inverting the tube<br />
*It was centrifuged at 12000 rpm for 10 minutes<br />
*The supernatant was transferred to a spin column inside a washtube <br />
*It was centrifuged at 12000 rpm for a minute<br />
*The supernatant was discarded and 500 µl of Wash Buffer with ethanol (w10) were added to the column<br />
*It was incubated for one minute at room temperature<br />
*The column was centrifuged at 12000 rpm for 1 minute<br />
*The liquid from the washtube was discarded and the column was placed inside the tube<br />
*700 µl of Wash Buffer W9 with ethanol were added to the column<br />
*The column with the washtube was centrifuged at 12000 rpm for 1 minute<br />
*The liquid from the washtube was discarded<br />
*The column in the washtube was centrifuged at 12000 rpm for 1 minute<br />
*The liquid from the washtube was discarded<br />
*The column was placed inside an eppendorf tube of 1.5ml<br />
*75 µl of preheated TE Buffer were added at the center of the column<br />
*The TE Buffer was previously warmed in water bath at 65⁰c-70⁰c for 3 minutes<br />
*The column was incubated for 1 minute at room temperature<br />
*The column was centrifuged at 12000 rpm for 2 minutes<br />
*The eppendorf tube contains the purified plasmid<br />
<br />
<br />
----<br />
'''Experiment 4: Preparation of mediums for lactobacillus and E. Coli (July 17th, 2013)'''<br />
<br />
*15.3 g of MRS were weighted for 300 ml, the 2% (0.306 g.) of the MRS was used to do agar-agar<br />
*The autoclave started to heat <br />
*8.75 g of LB agar were weighted to dilute in 500 ml of distilled water<br />
*0.5005 g of LB broth were weighted to be diluted in 250 ml of distilled water<br />
*The 3 mediums were introduced in the autoclave<br />
*After 54 minutes the sterilization was complete <br />
*0.750 g of Kanamycin sulfate were measured and they were diluted in 15 ml of distilled water<br />
*1.5 g of Ampicillium sodium were weighted and diluted in 15 ml<br />
*The mediums were collocated in the laminar flow cabinet to pour into the petri dishes and striate <br />
*The mediums never solidified <br />
*2% more of agar was added in the LB broth and the mediums were placed in the autoclave <br />
*The mediums were collocated in the laminar flow cabinet to be poured into the petri dishes and striate<br />
*But the mediums didn’t solidified<br />
<br />
<br />
----<br />
'''Experiment 5: Preparation of MRS agar mediums (July 19th, 2013)'''<br />
<br />
*The mediums didn’t solidify, because the agar added wasn’t enough<br />
*10.2 g of MRS broth were measured and 2 g of agar-agar were added<br />
*The pH of the mediums was 6.6 <br />
*A stock of HCl at 10% was added until the pH reached 6.4 <br />
*The mediums were sterilized with the autoclave for 15 minutes at 121°C <br />
*40 ml of agar were mixed with 40 µl of stock of ampicillin and they were poured into 2 petri dishes<br />
*40 ml of MRS agar were mixed with 40 µl of kanamycin stock and they were poured into 2 petri dishes <br />
*6 petri dishes were made with only MRS agar <br />
*2 control petri dishes with MRS agar were striated with L. Plantarum with 1.5 ml of glycerol stock <br />
*The petri dishes were incubated at 37°C for 2 hours<br />
<br />
<br />
----<br />
'''Experiment 6: Growth of E. Coli (July 21st, 2013)'''<br />
<br />
*No growth was discernible in the petri dishes with E. Coli, which were left incubating. This attributed to the use of an E. Coli sample, which was in refrigeration since 2011<br />
*The LB Agar medium was heated in an electric stove for approximately 10 minutes<br />
*A new sample of lyophilized E. Coli sample was opened and a representative sample was extracted to be rehydrated with LB broth<br />
*Liquid LB agar was poured into 2 petri dishes and they were left to solidify<br />
*Both dishes were striated with the rehydrated E. Coli<br />
*Both dishes were left incubating, after being sealed, during 24 hours at 37⁰c<br />
<br />
<br />
----<br />
'''Experiment 7: Glycerol stocks reactivation (July 22nd, 2013)'''<br />
<br />
*No discernible growth was observed in the L. Plantarum plates after a 72 hours incubation period. After consulting an external source, it was determined that this was due to glycerol stocks used, which weren’t reactivated<br />
*Two stocks of glycerol were reactivated with MRS broth<br />
*10 ml of MRS broth were added to a 1 ml stock in a falcon tube<br />
*The second stock was poured into another falcon tube, the result was 1.5 ml of stock with 15 ml of MRS broth<br />
*Both falcon tubes were sealed and left incubating at 37⁰c<br />
<br />
<br />
----<br />
'''Experiment 8: Overnight growth of E. Coli in LB broth (July 23rd, 2013)''' <br />
<br />
*5 ml of LB broth were poured into 2 falcon tubes each <br />
*An E. Coli colony was added to each tube with broth <br />
*Both tubes were sealed and left incubating at 37⁰c<br />
<br />
<br />
----<br />
'''Experiment 9: Preparation of mediums for lactobacillus and E. Coli (July 24th, 2013)'''<br />
<br />
*15.3 g of MRS and 6 g of bacto agar were weighted to be diluted in 300 ml<br />
*17.5 g of LB agar were weighted to prepare 500 ml<br />
*The MRS and LB agar were autoclaved <br />
*1 ml of E. Coli was inoculated in 100 ml of LB agar in a flask of 250 ml<br />
*5 g of LB broth were weighted<br />
*It was autoclaved for 15 minutes at 121⁰c<br />
<br />
<br />
----<br />
'''Experiment 10: Preparation of TE buffer (July 26th, 2013)'''<br />
<br />
*6.05 g of TRIS were weighted and diluted in 60 ml of distilled water<br />
*9.3041 g of EDTA were weighted and diluted in 60 ml of distilled water<br />
*The TRIS’ pH was 10.29, therefore HCl was added until the pH reached 8.3<br />
*The EDTA’s pH was 3.25, therefore NaOH crystals were added until the pH reached 7.79<br />
*They were sealed and autoclaved for 15 minutes at 121⁰c<br />
<br />
<br />
----<br />
'''Experiment 11: Preparation of competent E. Coli cells (July 28th, 2013)'''<br />
<br />
*A falcon tube was prepared with 50 ml of CaCl2 0.1M<br />
*Another falcon tube was prepared with CaCl2 0.1M/ 15% glycerol<br />
*4 eppendorf tubes of 1.5 ml were filled <br />
*The 4 tubes were left in ice for 10 minutes<br />
*The 4 tubes were centrifuged for 3 minutes at 6000 rpm and the supernatant was discarded<br />
*1.5 ml of the aforementioned culture were added to each tube<br />
*The tubes were centrifuged for 3 minutes at 6000 rpm <br />
*The supernatant was discarded<br />
*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M for each tube<br />
*The 4 tubes were incubated in ice for 20 minutes<br />
*The tubes were centrifuged again for 3 minutes at 6000 rpm<br />
*The supernatant was discarded<br />
*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each<br />
*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c<br />
<br />
<br />
----<br />
'''Experiment 12: L. Plantarum plasmid purification (July 28th, 2013)'''<br />
<br />
*1.5 ml of L. Plantarum culture in MRS broth were poured into 2 eppendorf tubes, 1.5 ml each<br />
*Both tubes were centrifuged for 4 minutes at 12000 rpm<br />
*The supernatant was discarded and another 1.5 ml of culture were added <br />
*Both tubes were centrifuged for 4 minutes at 12000 rpm<br />
*The supernatant was discarded <br />
*250 µl of resuspension buffer (R3) were added and the pellets were resuspended until the mixture was homogenous <br />
*The tubes were incubated at room temperature for 15 minutes<br />
*250 µl of Lysis buffer (L7) were added to each tube. They were gently mixed<br />
*The tubes were incubated at room temperature for 5 minutes<br />
*350 µl of precipitation buffer (N4) were added <br />
*Both tubes were vigorously mixed until a homogenous mixture was obtained <br />
*The tubes were centrifuged at 12000 rpm for 10 minutes<br />
*The supernatant was poured into a spin column in a wash tube of 2 ml <br />
*The columns were centrifuged at 12000 rpm for 1 minute <br />
*The supernatant was discarded and the column was placed in the new washtube<br />
*500 µl of wash buffer (W10) with ethanol were added to the column <br />
*The columns were incubated for 1 minute at room temperature<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The liquid that went through the columns was discarded<br />
*700 µl of wash buffer (W9) were added<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The liquid that went through the columns was discarded<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The liquid that went through the columns and the washtube was discarded<br />
*The columns were placed in eppendorf tubes of 1.5 ml<br />
*70 µl of TE buffer (previously heated at 65⁰c for 3 minutes) were added in the center of each column<br />
*The columns were incubated at room temperature for 1 minute <br />
*The columns were centrifuged at 12000 rpm for 2 minutes<br />
*The eppendorf tubes contain the purified plasmids<br />
*The columns were removed, the eppendorf tubes were sealed and they were refrigerated at 4⁰c until the next day<br />
<br />
<br />
----<br />
'''Experiment 13: Electrophoresis gel for L. Plantarum (July 29th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution <br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
<br />
Preparation of the sample to be placed in the gel<br />
*10 µl of each sample of purified plasmid were placed in eppendorf tubes of 1.5 ml<br />
*5 µl of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample<br />
<br />
Running the gel<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*15 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 15 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*No sample presented marks in the gel, only the gene ruler<br />
<br />
<br />
----<br />
'''Experiment 14: Overnight culture of L. Plnatarum and agarose preparation (July 29th, 2013)'''<br />
<br />
*2 colonies with one day of incubation were inoculated in MRS broth out of a 10 ml sample<br />
*They were placed in falcon tubes and left incubating at 37.2⁰c<br />
*35 ml of a TAE 1x solution were used to prepare agarose gel for electrophoresis. 0.35 g of agarose were added to the solution to solidify it<br />
*The liquid gel was poured into a casting tray and the well combs were placed<br />
<br />
<br />
----<br />
'''Experiment 15: Rehydration of plasmids in kit plates (July 30th, 2013)'''<br />
<br />
*The plate kits were numbered as indicated in the iGEM’s parts registry and the plasmids to be used were localized<br />
*The protective layer was pierced with a micro-pipet<br />
*Each plasmid was rehydrated with 10 µl of distilled water<br />
*The liquid was pipetted until it was homogeneous <br />
*It was left resting for 5 minutes to ensure its rehydration<br />
<br />
<br />
----<br />
'''Experiment 16: Electrophoresis gel for kit plates plasmids (July 30th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to form a 400 ml solution<br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
*The agarose with TAE 1x was heated in the microwave for 4 seconds<br />
Preparation of the sample to be run in gel<br />
*5 µl of each rehydrated plasmid were placed in eppendorf tubes <br />
*10 µl of 6x DNA Loading Dye were added and it was pipetted until the mixture was homogeneous<br />
Running the gel<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*15 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 15 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*Thin marks can be appreciated in samples psB4A5 and psB1A7<br />
<br />
<br />
----<br />
'''Experiment 17: E. Coli transformation with linearized plasmids (psB4k5, psB1k3, psB2k3) (July 31st, 2013)'''<br />
<br />
*The competent cells were taken out of deep freeze and were left to defrost in ice<br />
*They were mixed softly and 50 µl of competent cells were placed in a cold eppendorf tube <br />
*10 µl of linearized plasmid were added (psB4k5)<br />
*It was mixed gently and left incubating in ice for 30 minutes<br />
*The tube was put through heat shock in water at 42⁰c for 45 seconds<br />
*The tube was placed in ice for 2-5 minutes<br />
*900 µl of S.O.C. were added<br />
*The tube was left incubating for 1 hour at 37⁰c and 200 rpm<br />
*A dish with LB agar and kanamycin was striated with the transformed E. Coli<br />
*The dish was sealed and left incubating at 37⁰c<br />
*The same procedure was followed for plasmids psB1k3 and psB2k3<br />
<br />
<br />
----<br />
'''Experiment 18: Preparation of transformation buffer CaCl2 50mM (August 2nd, 2013)'''<br />
<br />
*1 ml of CaCl2 buffer 200mM were placed in a beaker<br />
*3 ml of sterile distilled water were added<br />
*With a syringe, it was filled to its maximum capacity with the buffer<br />
*A ministart 0.2µm filter was connected to the syringe<br />
*The content of the syringe was poured into a sterile falcon tube through the tube <br />
*The tube was left cooling in ice<br />
<br />
<br />
----<br />
'''Experiment 19: E. Coli transformation with pGLO (GFP) (August 3rd, 2013)'''<br />
<br />
*The competent cells were taken out of deep freeze and left defrosting in ice<br />
*They were gently mixed and 50 µl were placed in a cold 1.5 ml eppendorf tube<br />
*10 µl of pGLO were added<br />
*They were gently mixed and left incubating in ice for 30 minutes<br />
*The tube went through heat shock in water at 42⁰c for 45 seconds<br />
*The tube was left in ice for 5 minutes<br />
*900 µl of S.O.C. was added to the tube<br />
*It was gently mixed and left incubating for 1 hour at 37⁰c and 150 rpm <br />
*A control dish and a dish with antibiotic were striated <br />
*Both dishes were sealed and left incubating facing down at 37⁰c<br />
<br />
<br />
----<br />
'''Experiment 20: MRS and LB mediums preparation (August 5th, 2013)'''<br />
<br />
*15.3 g of MRS and 6 g of bacto agar were weighted to be diluted in 300 ml<br />
*17.5 g of LB agar were weighted to prepare 500 ml<br />
*The MRS and LB agar were autoclaved for 15 minutes at 121⁰c<br />
*They were left in refrigeration<br />
Results:<br />
*After an incubating period of 24 hours both, the control and the L-arabinose and ampicillin, plates were observed. Both plates presented bacterial growth. The bacteria in the L-arabinose plate were analyzed in the transilluminator and they glowed. Therefore, the transformation was successful and the gen was expressed correctly. Likewise, it was demonstrated that the previously prepared competent cells are effective. <br />
<br />
<br />
----<br />
'''Experiment 21: Preparation of plates with LB agar and antibiotic (August 6th, 2013)'''<br />
<br />
*The LB agar was heated in the electrical stove until it was completely liquefied <br />
*The next plates were prepared:<br />
**1 control (only agar)<br />
**2 with kanamycin<br />
**1 with kanamycin and L-arabinose<br />
**1 with ampicillin<br />
*Plate with kanamycin<br />
**40 µl of kanamycin and 40 ml of LB agar were poured into a beaker (for 2 dishes)<br />
**They were mixed and poured into 2 dishes<br />
*Plate with kanamycin and L-arabinose<br />
**20 µl of kanamycin, 600 µl of L-arabinose and 20 ml of LB agar were mixed in a beaker<br />
**The solution was poured into a petri dish<br />
*Plate with ampicillin<br />
**20 µl of ampicillin and 20 ml of agar were mixed in a beaker<br />
**The solution was poured into a dish<br />
*The mediums were prepared again to duplicate the amount of dishes<br />
*The plates solidified and they were properly labeled<br />
*A total of 9 dishes were prepared<br />
**1 with LB agar<br />
**4 with kanamycin<br />
**2 with kanamycin and L-arabinose<br />
**2 with ampicillin<br />
<br />
<br />
----<br />
'''Experiment 22: E. Coli transformation with linearized plasmids (psB4k5, psB1A7, psB2k3) (August 7th, 2013)'''<br />
<br />
*The DNA of each plasmid was rehydrated with 10 µl of sterile distilled water<br />
*Each well of the kit plates was pierced with a micro pipet<br />
*Each rehydrated DNA was stored in eppendorf tubes<br />
*The competent cells were taken out of deep freeze and were left to defrost in ice<br />
*The competent cells were mixed softly and 50 µl of competent cells were placed in a cold eppendorf tube <br />
*2 µl of linearized plasmid were added and pipetted until it was homogeneous <br />
*It was sealed and left incubating in ice for 30 minutes<br />
*The tube was put through heat shock in water at 42⁰c for 60 seconds<br />
*The tube was placed in ice for 5 minutes<br />
*200 µl of S.O.C. were added<br />
*The tube was left incubating for 2 hours at 37⁰c<br />
*(This was done with each plasmid)<br />
*The samples were striated with a Digralsky spreader in dishes with their correspondent antibiotic and/or inducer<br />
*For each plasmid it was inoculated:<br />
##In a dish, 20µl of a sample of transformed E. Coli (with antibiotics)<br />
##In a dish, 100 µl of the same transformed E. Coli (with antibiotics)<br />
##In a control dish, the rest of the transformed cells<br />
*they were sealed and left incubating at 37⁰c<br />
<br />
<br />
----<br />
'''Experiment 23: Preparation of competent cells with Lactobacillus Plantarum (August 8th, 2013)'''<br />
<br />
*2 eppendorf tubes of 1.5 ml were filled with L. Plantarum in MRS broth<br />
*The tubes were left in ice for 10 minutes<br />
*The tubes were centrifuged for 3 minutes at 6000 rpm and the supernatant was discarded<br />
*1.5 ml of the aforementioned culture were added to each tube<br />
*The tubes were centrifuged for 3 minutes at 6000 rpm <br />
*The supernatant was discarded<br />
*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M for each tube<br />
*The tubes were incubated in ice for 20 minutes<br />
*The tubes were centrifuged again for 3 minutes at 6000 rpm<br />
*The supernatant was discarded<br />
*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each<br />
*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c<br />
<br />
<br />
----<br />
'''Experiment 24: Purification of L. Plantarum plasmids (August 9th, 2013)'''<br />
<br />
*2 different samples of L. Plantarum were used, one from an isolated colony and another form a reactivated stock<br />
*1.5 ml of each sample were poured into eppendorf tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*Another 1.5 ml of the samples were added to the tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixtures were homogenous <br />
*The tubes were incubated for 15 minutes<br />
*250 µl of Lysis buffer (L7) were added to each tube and mixed gently<br />
*They were left incubating for 5 minutes<br />
*350 µl of precipitation buffer were added and it was vigorously shaken<br />
*Both tubes were centrifuged at 12000 rpm for 10 minutes<br />
*It was observed that before centrifuging the samples, the tube with the reactivated cells had a much larger volume than the other, which meant a mistake was probably made when pipetting <br />
*The supernatant was transferred to a spin column in a 2 ml washtube <br />
*The tubes were centrifuged for 1 minute at 12000 rpm<br />
*The liquid that passed through was discarded and the column was placed again in the washtube<br />
*500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute<br />
*Both tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded<br />
*700 µl of Wash buffer (W9) were added<br />
*Both columns were centrifuged at 12000 rpm for 1 minute<br />
*The supernatant was discarded and the columns were transferred to eppendorf tubes of 1.5 ml <br />
*70 µl of TE buffer were added to each tube (previously heated at 65⁰c for 3 minutes)<br />
*The tubes were incubated for 1 minute at room temperature<br />
*Both tubes were centrifuged at 12000 rpm for 2 minutes<br />
*The columns were discarded and the tubes were sealed and refrigerated at 4⁰c<br />
<br />
<br />
----<br />
'''Experiment 25: Gel for electrophoresis of plasmids (August 9th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution <br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
*The agarose with the gel was heated in the microwave for 45 seconds<br />
Preparation of the sample to be placed in the gel<br />
*10 µl of each sample of purified plasmid were placed in eppendorf tubes of 1.5 ml<br />
*5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample<br />
Running the gel<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*8 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 15 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*Very soft marks were observable for the gene ruler and the L. Plantarum sample<br />
<br />
<br />
----<br />
'''Experiment 26: Preparation of dishes with antibiotics (August 10th, 2013)'''<br />
<br />
*The LB agar was heated in the electrical stove until it was completely liquefied <br />
*The next plates were prepared:<br />
**4 control (agar only)<br />
**4 with kanamycin<br />
**2 with kanamycin and L-arabinose<br />
**2 with ampicillin<br />
*Plate with kanamycin<br />
**80 µl of kanamycin and 80 ml of LB agar were poured into a beaker (for 2 dishes)<br />
**They were mixed and poured into 4 dishes<br />
*Plate with kanamycin and L-arabinose<br />
**40 µl of kanamycin, 1200 µl of L-arabinose and 40 ml of LB agar were mixed in a beaker<br />
**The solution was poured into 2 petri dishes<br />
*Plate with ampicillin<br />
**40 µl of ampicillin and 40 ml of agar were mixed in a beaker<br />
**The solution was poured into 2 dishes<br />
*The mediums were prepared again to duplicate the amount of dishes<br />
*The plates solidified, properly labeled and refrigerated<br />
<br />
<br />
----<br />
'''Experiment 27: Transformation of E. Coli with linearized plasmids (psB4k5, psB1A7, psB2k3) (August 10th, 2013)'''<br />
<br />
*The competent cells were taken out of deep freeze and were left to defrost in ice<br />
*The competent cells were mixed softly and 50 µl of competent cells were placed in a cold eppendorf tube <br />
*2 µl of linearized plasmid were added and pipetted until it was homogeneous <br />
*It was sealed and left incubating in ice for 30 minutes<br />
*The tube was put through heat shock in water at 42⁰c for 60 seconds<br />
*The tube was placed in ice for 5 minutes<br />
*200 µl of S.O.C. were added<br />
*The tube was left incubating for 2 hours at 37⁰c and 150 rpm<br />
*(This was done with each plasmid)<br />
*Each plasmid was striated in a plate<br />
*They were sealed and left incubating at 37⁰c<br />
Results:<br />
*After an incubating period of 60 hours the plates with transformed E. Coli, plates were observed. Both plates presented bacterial growth. Therefore, the transformation was successful. Likewise, it was demonstrated that the previously prepared competent cells are effective. However, one dish presented a fungus, which was due to antibiotic which wasn’t filtered. Besides, the plates are not expressing GFP nor RFP<br />
<br />
<br />
----<br />
'''Experiment 28: Preparation of LB broth (August 12th, 2013)'''<br />
<br />
*6.0034 g of LB broth were weighted and diluted in 250 ml of distilled water. Afterwards, another 50 ml were added<br />
*A cotton and Kraft paper cap was used to cover the flask, and it was autoclaved for 15 minutes at 121⁰c<br />
<br />
<br />
----<br />
'''Experiment 29: Preparation of antibiotics (August 13th, 2013)'''<br />
<br />
*1.5 g of ampicillin were diluted in 15 ml of sterile water<br />
*0.750 g of kanamycin were weighted and diluted in 15 ml of sterile water<br />
*A syringe was used to its maximum capacity to hold the ampicillin stock<br />
*A Ministart hydrophilic filter 0.20µm was attached to the syringe<br />
*The content of the syringe was filtered into a falcon tube <br />
*The same procedure was used for the kanamycin stock<br />
*The stocks were sealed and stored in the refrigerator<br />
<br />
<br />
----<br />
'''Experiment 30: Purification of L. Plantarum plasmids (August 15th, 2013)'''<br />
<br />
*A sample from an isolated colony was used<br />
*1.5 ml of the sample was poured into an eppendorf tube<br />
*The tube was centrifuged for 4 minutes at 12000 rpm and the pellet was discarded<br />
*Another 1.5 ml of the samples were added to the tube<br />
*The tube was centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*250 µl of resuspension buffer (R3) were added and the sample was pipetted until the mixture was homogenous <br />
*The tube was incubated for 15 minutes<br />
*250 µl of Lysis buffer (L7) was added to the tube and mixed gently<br />
*It was left incubating for 5 minutes<br />
*350 µl of precipitation buffer were added and it was vigorously shaken<br />
*The tube was centrifuged at 12000 rpm for 10 minutes<br />
*The supernatant was transferred to a spin column in a 2 ml washtube <br />
*The tube was centrifuged for 1 minute at 12000 rpm<br />
*The liquid that passed through was discarded and the column was placed again in the washtube<br />
*500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute<br />
*The tube was centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded<br />
*700 µl of Wash buffer (W9) were added<br />
*The column was centrifuged at 12000 rpm for 1 minute<br />
*The supernatant was discarded and the column was transferred to an eppendorf tube of 1.5 ml <br />
*70 µl of TE buffer were added to the tube (previously heated at 65⁰c for 3 minutes)<br />
*The tube was incubated for 1 minute at room temperature<br />
*The tube was centrifuged at 12000 rpm for 2 minutes<br />
*The columns were discarded and the tubes were sealed and refrigerated at 4⁰c<br />
<br />
<br />
----<br />
'''Experiment 31: Gel for electrophoresis of L. Plantarum (purified plasmids) (August 15th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution <br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
*The agarose with the gel was heated in the microwave for 45 seconds<br />
Preparation of the sample to be placed in the gel<br />
*10 µl of each sample of purified plasmids were placed in eppendorf tubes of 1.5 ml<br />
*5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample<br />
Running the gel<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*15 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 20 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*There was no discernible mark other than the gene ruler <br />
<br />
<br />
----<br />
'''Experiment 32: Purification of L. Plantarum plasmids (August 15th, 2013)'''<br />
<br />
*A sample from an isolated colony was used<br />
*1.5 ml of the sample was poured into an eppendorf tube, a duplicate was made<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the pellet was discarded<br />
*Another 1.5 ml of the samples were added to the tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixture was homogenous <br />
*The tubes were incubated for 15 minutes<br />
*250 µl of Lysis buffer (L7) were added to the tubes and mixed gently<br />
*They were left incubating for 5 minutes<br />
*350 µl of precipitation buffer were added and it was vigorously shaken<br />
*The tubes were centrifuged at 12000 rpm for 10 minutes<br />
*The supernatant was transferred to a spin column in a 2 ml washtube <br />
*The tubes were centrifuged for 1 minute at 12000 rpm<br />
*The liquid that passed through was discarded and the column was placed again in the washtube<br />
*500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute<br />
*The tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded<br />
*700 µl of Wash buffer (W9) were added<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The supernatant was discarded and the columns were transferred to an eppendorf tube of 1.5 ml <br />
*70 µl of TE buffer were added to the tube (previously heated at 65⁰c for 3 minutes)<br />
*The tube was incubated for 11 minute at room temperature (because the centrifuge was occupied)<br />
*The tube was centrifuged at 12000 rpm for 2 minutes<br />
*The columns were discarded and the tubes were sealed and refrigerated at 4⁰c<br />
<br />
<br />
----<br />
'''Experiment 33: Purification of transformed E. Coli plasmids (psB3k5, psB4k5, psB1A7, psB2k3) (August 17th, 2013)'''<br />
<br />
*4 samples E. coli from an isolated colony were used<br />
*1.5 ml of each sample were poured into eppendorf tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the pellet was discarded<br />
*Another 1.5 ml of the samples were added to the tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixtures were homogenous <br />
*The tubes were incubated for 15 minutes<br />
*250 µl of Lysis buffer (L7) were added to each tube and mixed gently<br />
*They were left incubating for 5 minutes<br />
*350 µl of precipitation buffer were added and it was vigorously shaken<br />
*The tubes were centrifuged at 12000 rpm for 10 minutes<br />
*The supernatant was transferred to a spin column in a 2 ml washtube <br />
*The tubes were centrifuged for 1 minute at 12000 rpm<br />
*The liquid that passed through was discarded and the column was placed again in the washtube<br />
*500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute<br />
*Both tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded<br />
*700 µl of Wash buffer (W9) were added<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The supernatant was discarded and the columns were transferred to eppendorf tubes of 1.5 ml <br />
*70 µl of TE buffer were added to each tube (previously heated at 65⁰c for 3 minutes)<br />
*The tubes were incubated for 1 minute at room temperature<br />
*Both tubes were centrifuged at 12000 rpm for 2 minutes<br />
*The columns were discarded and the tubes were sealed and refrigerated at 4⁰c<br />
<br />
<br />
----<br />
'''Experiment 34: Gel for electrophoresis of L. Plantarum plasmids and plasmids (psB3k5, psB4k5, psB1A7, psB2k3) (August 17th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution <br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
*The agarose with the gel was heated in the microwave for 45 seconds<br />
Preparation of the sample to be placed in the gel<br />
*10 µl of each sample of purified plasmids were placed in eppendorf tubes of 1.5 ml<br />
*5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample<br />
Running the gel<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*15 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 20 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*There are marks in the gel; however most of the samples are blurred. The best defined marks are in track 7, at kbs. These marks are due to a faulty purification<br />
<br />
<br />
----<br />
'''Experiment 35: Purification of L. Plantarum plasmids and transformed E. Coli plasmids (psB3k5, psB4k5, psB1A7, psB2k3, psB1A7) (August 18th, 2013)'''<br />
<br />
*A sample of broth with L. Plantarum and LB with E. Coli was used for each corresponding plasmid<br />
*1.5 ml of each sample were poured into eppendorf tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the pellet was discarded<br />
*Another 1.5 ml of the samples were added to the tubes<br />
*The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded<br />
*250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixtures were homogenous <br />
*The tubes were incubated for 15 minutes<br />
*250 µl of Lysis buffer (L7) were added to each tube and mixed gently<br />
*They were left incubating for 5 minutes<br />
*350 µl of precipitation buffer were added and it was vigorously shaken<br />
*The tubes were centrifuged at 12000 rpm for 10 minutes<br />
*There was debris floating, so the supernatant was transferred to another tube and it was centrifuged again at 12000 rpm for 10 minutes<br />
*The supernatant was transferred to a spin column in a 2 ml washtube <br />
*The tubes were centrifuged for 1 minute at 12000 rpm<br />
*The liquid that passed through was discarded and the column was placed again in the washtube<br />
*500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute<br />
*Both tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded<br />
*700 µl of Wash buffer (W9) were added<br />
*The columns were centrifuged at 12000 rpm for 1 minute<br />
*The supernatant was discarded and the columns were transferred to eppendorf tubes of 1.5 ml <br />
*70 µl of TE buffer were added to each tube (previously heated at 65⁰c for 3 minutes)<br />
*The tubes were incubated for 1 minute at room temperature<br />
*Both tubes were centrifuged at 12000 rpm for 2 minutes<br />
*The columns were discarded and the tubes were sealed and refrigerated at 4⁰c<br />
<br />
<br />
----<br />
'''Experiment 36: Gel for electrophoresis of purified plasmids (August 18th, 2013)'''<br />
<br />
Agarose preparation<br />
*TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution <br />
*A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose) <br />
*The agarose with the gel was heated in the microwave for 45 seconds<br />
Preparation of the sample to be placed in the gel<br />
*10 µl of each sample of purified plasmids were placed in eppendorf tubes of 1.5 ml<br />
*5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample<br />
Running the gel (x2, because 2 gels were made)<br />
*The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs<br />
*It solidified and the well combs were removed <br />
*The gel was placed in the gel box, with the wells on the side of the anode <br />
*The box was filled with TAE 1x up to the indicated measure<br />
*15 µl of gene ruler were placed in the first well <br />
*15 µl of each sample were placed in consecutive wells <br />
*The electrophoresis box was closed and the power line was connected <br />
*The current was set to 100 V and it was left running for 55 minutes<br />
*The current was turned off and the gel was removed from the chamber<br />
*The gel was left rocking in Ethidium bromide for 20 minutes<br />
*The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator<br />
*The results were observed with UV light<br />
*Both gels presented intense marks in the tracks correspondent to the purified plasmids of E. Coli in the line of 20 kbs. The gels of L. Plantarum showed no marks<br />
<br />
<br />
----<br />
'''Experiment 37: Preparation of MRS broth, preparation of glycerol stocks and reactivation of glycerol stocks (August 19th, 2013)'''<br />
<br />
*10.2 g of MRS broth were weighted<br />
*The MRS broth was diluted in 200 ml of distilled water<br />
*The mediums were autoclaved for 15 min at 121⁰c<br />
*15 ml of MRS broth and 15 ml of glycerol (100%) were mixed in a 45 ml falcon tube, in the laminar flow cabinet<br />
*An aliquot of L. Plantarum was added to the solution and it was sealed with parafilm<br />
*1 glycerol stock was reactivated with MRS broth <br />
*A 1.5 ml stock was used with 15 ml of MRS broth, mixed in a falcon tube<br />
*Both tubes were left incubating at 37⁰c and 160 rpm<br />
<br />
<br />
----<br />
'''Experiment 38: Reactivation of the new L. Plantarum strain with glycerol stock (August 19th, 2013)'''<br />
<br />
*15 ml of MRS broth were mixed with 15 ml of glycerol (100%) in a 45 ml falcon tube<br />
*An aliquot of the new L. Plantarum was added and it was sealed with parafilm<br />
<br />
<br />
----<br />
'''E. Coli transformation with promoter 1,2 and rbs '''<br />
<br />
*Take the competent cells out of deep freeze and let them defrost in ice<br />
*Gently mix and put 50 µl in a cold 1.5 ml eppendorf tube<br />
*Add 10 µl of pGLO <br />
* Gently mix them and leave them incubating in ice for 30 minutes<br />
*Cause the tube to go through heat shock, placing it in water at 42⁰c for 45 seconds<br />
*Cool the tube in ice for 5 minutes<br />
*Add 900 µl of S.O.C. to the tube<br />
*Gently mix it and leave it incubating for 1 hour at 37⁰c and 150 rpm <br />
*Striate a control dish and a dish with antibiotic <br />
*Seal both dishes and leave them incubating facing down at 37⁰c</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ProtocolsTeam:BIOSINT Mexico/Protocols2013-09-28T04:02:24Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Protocols</h1> </html><br />
----<br />
----<br />
'''Preparation of growth mediums (MRS and LB)''' <br />
<br />
*Weight 18.6 g of Agar MRS to prepare 300ml of medium<br />
*Weight 7 g of Agar LB to prepare 200ml of medium<br />
*Dilute 5 g of A-MRS in 80 ml of distilled water<br />
*Add 5 g and 170 ml of distilled water <br />
*Dilute 7 g of Agar LB in 100ml of distilled water<br />
*Add 100ml of distilled water <br />
*Place the autoclave tape<br />
*Set the autoclave<br />
*When the temperature of the autoclave reaches 120⁰c, cut the power by half<br />
*When the temperature reaches 105⁰c, open the valve. <br />
*Retrieve the mediums<br />
<br />
<br />
----<br />
'''Preparation of MRS broth and glycerol stocks'''<br />
<br />
*Weight 0.7679 g of powder for MRS broth <br />
*Measure 15ml of distilled water in a 100ml measuring cylinder <br />
*Weight 10.009g of powder for MRS broth <br />
*Dilute it in 100ml of distilled water<br />
*Add 100ml of distilled water <br />
*Dilute 0.7679g of powder for MRS broth in 15ml of distilled water<br />
*Mark the flasks with autoclave tape and sterilize them for 15 minutes at 121⁰c<br />
*Label the broth <br />
*Mix the 15ml of MRS broth and the 15ml of glycerol to 100% in the laminar flow cabinet. Shake them gently until a homogenous mixture is obtained in a falcon tube of 45ml<br />
*Add an aliquot of L. Plantarum to the broth and glycerol solution and seal it with parafilm<br />
*Incubate it at 37⁰c and 200rpm<br />
*After 24 hours, take the sample out of the incubator to striate it<br />
*Place 2500 (x2)ml in 22 eppendorf tubes and using a micropipette of 200µl , cultivate and recompile the mixture of L. Plantarum contained in the falcon tube <br />
*Centrifuge 1 eppendorf tube at 13.4 rpm for 3 minutes <br />
*Discard the supernatant and add a solution of MRS broth (500ml) <br />
*Once the mixture is made, striate two petri dishes with MRS Agar in the laminar flow cabinet<br />
*Place the petri dishes facing down in the incubator at 37⁰c and 200 rpm<br />
*Store the remaining samples (21 falcon tubes) in deep freezing <br />
*Striate E. Coli K12 in two petri dishes with LB Agar <br />
*Place the dishes in the incubator at 37⁰c.<br />
<br />
<br />
----<br />
'''Plasmid purification'''<br />
<br />
*Centrifuge an eppendorf tube of 1.5ml with L. Plantarum culture at 12000 rpm for 1 minute, two times<br />
*Discard the supernatant and resuspend the tube’s content with more L. Plantarum culture in MRS broth<br />
*Centrifuge the eppendorf tube for 15min at 6000 rpm<br />
*Discard the supernatant <br />
*Resuspend the pellet in 250ml resuspension buffer<br />
*Add 250ml of Lysis Buffer L7<br />
*Gently mix the tube carefully inverting it 5 times carefully<br />
*Add and mix softly 350ml of precipitation Buffer N4, inverting the tube<br />
*Centrifuge it at 12000 rpm for 10 minutes<br />
*Transfer the supernatant into a spin column inside a washtube <br />
*Centrifuge it at 12000 rpm for a minute<br />
*Discard the supernatant and add 500 ml of Wash Buffer with ethanol (w10) to the column<br />
*Incubate it for one minute at room temperature<br />
*Centrifuge the column at 12000 rpm for 1 minute<br />
*Discard the liquid from the washtube and place the column inside the tube<br />
*Add 700ml of Wash Buffer W9 with ethanol to the column<br />
*Centrifuge the column with the washtube at 12000 rpm for 1 minute<br />
*Discard the liquid from the washtube<br />
*Centrifuge the column with the washtube at 12000 rpm for 1 minute<br />
*Discard the liquid from the washtube<br />
*Place the column inside an eppendorf tube of 1.5ml<br />
*Add 75ml of preheated TE Buffer at the center of the column<br />
*(Warm the TE Buffer previously in water bath at 65⁰c-70⁰c for 3 minutes)<br />
*Incubate the column for 1 minute at room temperature<br />
*The column was centrifuged at 12000 rpm for 2 minutes<br />
*(The eppendorf tube contains the purified plasmid)<br />
<br />
<br />
----<br />
'''Preparation of TE buffer'''<br />
<br />
*Dilute 6.05 g of TRIS in 60 ml of distilled water<br />
*Dilute 9.3041 g of EDTA in 60 ml of distilled water<br />
*Add HCl until the TRIS’ pH reaches 8.3<br />
*Add NaOH crystals until the EDTA’S pH reaches 7.79<br />
*Seal and autoclave for 15 minutes at 121⁰c<br />
<br />
<br />
----<br />
'''Preparation of competent E. Coli cells'''<br />
<br />
*Prepare a falcon tube with 50 ml of CaCl2 0.1M<br />
*Prepare another falcon tube with CaCl2 0.1M/ 15% glycerol<br />
*Fill 4 eppendorf tubes of 1.5 ml with the E. Coli culture <br />
**2 of them with 1.5 ml each of E. Coli culture in LB broth (1)<br />
**2 of them with 1.5 ml each of E. Coli culture in LB broth (2)<br />
*Leave the 4 tubes in ice for 10 minutes<br />
*Centrifuge the 4 tubes for 3 minutes at 6000 rpm and discard the supernatant <br />
*Add 1.5 ml of the culture to each tube<br />
*Centrifuge the tubes for 3 minutes at 6000 rpm <br />
*Discard the supernatant <br />
*gently resuspend the pellet with 1.2 ml CaCl2 0.1M for each tube<br />
*Incubate the 4 tubes in ice for 20 minutes<br />
*Centrifuge the tubes for 3 minutes at 6000 rpm<br />
*Discard the supernatant <br />
*Resuspend the pellets with 0.6 ml of CaCl2 0.1M/ 15% glycerol each<br />
*Pour them into micro-tubes (300 µl/tube), seal them and freeze them at -80⁰c<br />
<br />
<br />
----<br />
'''Electrophoresis gel for L. Plantarum'''<br />
<br />
Agarose preparation<br />
*Prepare TAE 1x with 392 ml of distilled water and 8 ml of TAE 50x (for a 400 ml solution) <br />
*Add 0.35 g to 35 ml (grams of agarose) <br />
Preparation of the sample to be placed in the gel<br />
*Place 10 µl of each sample of purified plasmid in eppendorf tubes of 1.5 ml<br />
*Add 5 µl of 6x DNA Loading Dye to each one, pipetting until completely homogenizing the sample<br />
Running the gel<br />
*Pour the agarose mixture into the casting tray for 35 ml gels with their respective well combs<br />
*Remove the well combs once it has solidified <br />
*Place the gel in the gel box, with the wells on the side of the anode <br />
*Fill the box with TAE 1x up to the indicated measure (equal quantities on both sides of the box)<br />
*Pour 15 µl of gene ruler in the first well with a micro pipet<br />
*Pour 15 µl of each sample in consecutive wells with a micro pipet<br />
*Close the electrophoresis box and connect the power line <br />
*Set the current to 100 V and leave it running for 55 minutes<br />
*Turn off the current and remove the gel from the chamber<br />
*Leave the gel rocking in Ethidium bromide for 15 minutes (always use gloves when handling Ethidium bromide)<br />
*Take the gel out of the platform and of the Ethidium bromide and place it in the transiluminator<br />
*Observe the results with UV light<br />
<br />
<br />
----<br />
'''Rehydration of plasmids in kit plates'''<br />
<br />
*Number the plate kits as indicated in the iGEM’s parts registry and the plasmids to be used <br />
*Pierce the protective layer with a micro-pipet<br />
*Rehydrate each plasmid with 10 µl of distilled water<br />
*Pipet the liquid until it is homogeneous <br />
*It was left resting for 5 minutes to ensure its rehydration<br />
<br />
<br />
----<br />
'''Preparation of calcium chloride buffer for transformation 200mM'''<br />
<br />
*Sterilize distilled water in the autoclave for 15 minutes at 121⁰c<br />
*Dilute 1.1159 g of calcium chloride in 50 ml of sterile distilled water <br />
*Fill a syringe to its maximum capacity with the buffer<br />
*Connect a ministart 0.2µm filter to the syringe<br />
*Pour the content of the syringe into a sterile falcon tube through the filter<br />
*Seal it and place it in refrigeration<br />
<br />
<br />
----<br />
'''Preparation of transformation buffer CaCl2 50mM'''<br />
<br />
*Place 1 ml of CaCl2 buffer 200mM in a beaker<br />
*Add 3 ml of sterile distilled water were added<br />
*Fill a syringe to its maximum capacity with the buffer<br />
*Connect A ministart 0.2µm to the syringe<br />
*Pour the content of the syringe into a sterile falcon tube through the filter <br />
*Refrigerate<br />
<br />
<br />
----<br />
'''E. Coli transformation with pGLO (GFP)'''<br />
<br />
*Take the competent cells out of deep freeze and let them defrost in ice<br />
*Gently mix and put 50 µl in a cold 1.5 ml eppendorf tube<br />
*Add 10 µl of pGLO <br />
* Gently mix them and leave them incubating in ice for 30 minutes<br />
*Cause the tube to go through heat shock, placing it in water at 42⁰c for 45 seconds<br />
*Cool the tube in ice for 5 minutes<br />
*Add 900 µl of S.O.C. to the tube<br />
*Gently mix it and leave it incubating for 1 hour at 37⁰c and 150 rpm <br />
*Striate a control dish and a dish with antibiotic <br />
*Seal both dishes and leave them incubating facing down at 37⁰c<br />
<br />
<br />
----<br />
'''Preparation of plates with LB agar and antibiotic'''<br />
<br />
*Heat the LB agar was in the electrical stove until it is completely liquefied <br />
*The next plates were prepared:<br />
Plate with kanamycin<br />
*Pour 40 µl of kanamycin and 40 ml of LB agar into a beaker (for 2 dishes)<br />
*Mix them and pour them into 2 dishes<br />
Plate with kanamycin and L-arabinose<br />
*Mix 20 µl of kanamycin, 600 µl of L-arabinose and 20 ml of LB agar in a beaker<br />
*Pour the solution into a petri dish<br />
Plate with ampicillin<br />
*Mix 20 µl of ampicillin and 20 ml of agar in a beaker<br />
*Pour the solution into a dish<br />
*Let the plates solidify and label them<br />
<br />
----<br />
'''E. Coli transformation with parts promoter 1,2 and rbs '''<br />
<br />
*Take the competent cells out of deep freeze and let them defrost in ice<br />
*Gently mix and put 50 µl in a cold 1.5 ml eppendorf tube<br />
*Add 10 µl of pGLO <br />
* Gently mix them and leave them incubating in ice for 30 minutes<br />
*Cause the tube to go through heat shock, placing it in water at 42⁰c for 45 seconds<br />
*Cool the tube in ice for 5 minutes<br />
*Add 900 µl of S.O.C. to the tube<br />
*Gently mix it and leave it incubating for 1 hour at 37⁰c and 150 rpm <br />
*Striate a control dish and a dish with antibiotic <br />
*Seal both dishes and leave them incubating facing down at 37⁰c</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ConstructionTeam:BIOSINT Mexico/Construction2013-09-28T03:56:46Z<p>Gabyqz: /* Repressor switch */</p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
__NOTOC__<br />
<br />
----<br />
<html><h1 class="myowntitle">Construction</h1> </html><br />
<br />
===Repressor switch===<br />
----<br />
[[File:Repressorbio.jpg|center|300px]]<br />
<br />
<br />
:Based on the system of iGEM team Slovenia 2012, we adapted the repressor system using TAL A/KRAB. The objective is to reproduced a simplex circuit in a Lactobacillus chassis. <br />
<br />
:The main goal of our xenobiotics biosensor is to detect organochorides in different concentration in order to activated a correct answered.<br />
<br />
:High concentration: When the concentration is high Gal4 DBD could add to UAS and activated linA, a protein wich degrades pesticides, and at the same time express a repressor protein which inhibits the activity of the correpressor that controled the kill switch system. <br />
<br />
:Low concentration: When the concentration decrease, there is not enought UAS activity in the linA system so the repressor activity stops and the peptides that kill the bacteria starts to be produced.<br />
<br />
===Circuit===<br />
----<br />
[[File:Diapositiva1.JPG|center|700px]]</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/Future_researchTeam:BIOSINT Mexico/Future research2013-09-28T03:49:15Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Future research</h1> </html><br />
----<br />
----<br />
[[File:IMG_0134.JPG|left|450px]]<br />
<br />
<br />
<br />
:Probiotics exhibit different kinds of therapeutic activity in various diseases through the gastrointestinal mucosal immune regulation, or stimulation of the functions of the epithelial barrier. Probiotics represent a reasonable treatment indication for some clinical conditions, and in progress on the investigation of new preparations makes them a promising alternative treatment.<br />
<br />
:Along with other advances in the understanding of the molecular mechanisms involved in the therapeutic potential of the probiotics compounds, biomedical technological development has opened new perspectives for therapeutic research microbial strains genetically or synthetically processed, as well as modified systems to allow the arrival of probiotics to specific sites of action in the gut.<br />
<br />
:Our project opens the possibility of using recombinant strains employing synthetic vectors to promote the expression of microbial products with different traditional functions. In the future views of our project, it can adopt different functions to optimize controlled release of substances sensitive to degradation in the upper sections of the intestine. Suggesting the capacity beyond exposer this days, in the future will be possible through probiotics with these features induce biochemical, microbiological or histological, with a pretty potential clinical benefit in human disease.<br />
<br />
<br />
[[File:Investigation-lab-350.jpg]]<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
===Reference:===<br />
<br />
L, Menchén. (2013). Probiotics: Therapeutic applications in digestive pathologies. Gregorio Marañon Hospital. Department of Medicine. Health Research Institute. Madrid, España.</div>Gabyqzhttp://2013.igem.org/File:Investigation-lab-350.jpgFile:Investigation-lab-350.jpg2013-09-28T03:48:14Z<p>Gabyqz: </p>
<hr />
<div></div>Gabyqzhttp://2013.igem.org/File:GFP.jpgFile:GFP.jpg2013-09-28T03:46:52Z<p>Gabyqz: uploaded a new version of &quot;File:GFP.jpg&quot;</p>
<hr />
<div></div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ConstructionTeam:BIOSINT Mexico/Construction2013-09-28T03:46:47Z<p>Gabyqz: /* Repressor switch */</p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
__NOTOC__<br />
<br />
----<br />
<html><h1 class="myowntitle">Construction</h1> </html><br />
<br />
===Repressor switch===<br />
----<br />
[[File:Repressorbio.jpg|center|300px]]<br />
<br />
<br />
:Based on the system of iGEM team Slovenia 2012, we adapted de repressor system. The objective is to reproduced a simplex circuit in a Lactobacillus chassis. <br />
<br />
:The main goal of our xenobiotics biosensor is to detect organochorides in different concentration in order to activated a correct answered. When the concentration is high Gal4 DBD could add to UAS and activated linA, a protein wich degrades pesticides, and at the same time express a repressor protein wich inhibits the activity of the promoter that controled the kill switch system. When the concentration decrease, there is not enought UAS activity in the linA system so the peptides that kill the bacteria starts to be produced.<br />
<br />
===Circuit===<br />
----<br />
[[File:Diapositiva1.JPG|center|700px]]</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ConstructionTeam:BIOSINT Mexico/Construction2013-09-28T03:46:22Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
__NOTOC__<br />
<br />
----<br />
<html><h1 class="myowntitle">Construction</h1> </html><br />
<br />
===Repressor switch===<br />
----<br />
[[File:Repressorbio.jpg|center|300px]]<br />
<br />
<br />
Based on the system of iGEM team Slovenia 2012, we adapted de repressor system. The objective is to reproduced a simplex circuit in a Lactobacillus chassis. <br />
<br />
The main goal of our xenobiotics biosensor is to detect organochorides in different concentration in order to activated a correct answered. When the concentration is high Gal4 DBD could add to UAS and activated linA, a protein wich degrades pesticides, and at the same time express a repressor protein wich inhibits the activity of the promoter that controled the kill switch system. When the concentration decrease, there is not enought UAS activity in the linA system so the peptides that kill the bacteria starts to be produced. <br />
<br />
<br />
===Circuit===<br />
----<br />
[[File:Diapositiva1.JPG|center|700px]]</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ModelingTeam:BIOSINT Mexico/Modeling2013-09-28T03:45:19Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Modeling</h1> </html><br />
----<br />
----<br />
<br />
GFP tends to get fluorescents at the 395 nm because of the excitation of the protein this extend to the 475 nm where the green light is still visible, so we used this information in order to determine an equation that represents the exponential growth of the luminescent.<br />
What we pretend with this mathematic model is to represent in a numerical way, the expression of the protein in the cell according with the emission that each concentration of GFP provides. <br />
<br />
<br />
[[File:Tabla.jpg]]<br />
<br />
We used linear regression in order to get the values of the constants, using statistical methods that also are applied in the equation. The slope is the value given to b and the intersection is the value for a.<br />
<br />
<br />
y=a/(1+be^(-ct))<br />
<br />
be^(-ct)=a/y-1<br />
<br />
ln(b)-ct=ln(a/y)-ln(1)<br />
<br />
c=(ln)(b)-ln(a)-ln(y))/t<br />
<br />
<br />
[[File:Plot.jpg]]<br />
<br />
<br />
The graph represent an exponential growth of luminescent that at the same time is restricted the own GFP´s spectrum which only gives green color from 395nm to 475nm, also external factors like the medium in which bacteria growth because is limited.<br />
<br />
Recovered on september 5 from 2013 with an url of http://dwb.unl.edu/Teacher/NSF/C08/C08Links/pps99.cryst.bbk.ac.uk/projects/gmocz/gfp.htm</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ModelingTeam:BIOSINT Mexico/Modeling2013-09-28T03:42:57Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Modeling</h1> </html><br />
----<br />
----<br />
<br />
GFP tends to get fluorescents at the 395 nm because of the excitation of the protein this extend to the 475 nm where the green light is still visible, so we used this information in order to determine an equation that represents the exponential growth of the luminescent.<br />
What we pretend with this mathematic model is to represent in a numerical way, the expression of the protein in the cell according with the emission that each concentration of GFP provides. <br />
<br />
<br />
[[File:Tabla.jpg]]<br />
<br />
We used linear regression in order to get the values of the constants, using statistical methods that also are applied in the equation. The slope is the value given to b and the intersection is the value for a.<br />
<br />
<br />
y=a/(1+be^(-ct))<br />
<br />
be^(-ct)=a/y-1<br />
<br />
ln(b)-ct=ln(a/y)-ln(1)<br />
<br />
c=(ln)(b)-ln(a)-ln(y))/t<br />
<br />
<br />
[[File:Plot.jpg]]<br />
<br />
<br />
The graph represent an exponential growth of luminescent that at the same time is restricted the own GFP´s spectrum which only gives green color from 395nm to 475nm, also external factors like the medium in which bacteria growth because is limited.</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ModelingTeam:BIOSINT Mexico/Modeling2013-09-28T03:42:23Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Modeling</h1> </html><br />
----<br />
----<br />
<br />
GFP tends to get fluorescents at the 395 nm because of the excitation of the protein this extend to the 475 nm where the green light is still visible, so we used this information in order to determine an equation that represents the exponential growth of the luminescent.<br />
What we pretend with this mathematic model is to represent in a numerical way, the expression of the protein in the cell according with the emission that each concentration of GFP provides. <br />
<br />
<br />
y=a/(1+be^(-ct))<br />
<br />
[[File:Tabla.jpg]]<br />
<br />
We used linear regression in order to get the values of the constants, using statistical methods that also are applied in the equation. The slope is the value given to b and the intersection is the value for a.<br />
<br />
<br />
y=a/(1+be^(-ct))<br />
<br />
be^(-ct)=a/y-1<br />
<br />
ln(b)-ct=ln(a/y)-ln(1)<br />
<br />
c=(ln)(b)-ln(a)-ln(y))/t<br />
<br />
<br />
[[File:Plot.jpg]]<br />
<br />
<br />
The graph represent an exponential growth of luminescent that at the same time is restricted the own GFP´s spectrum which only gives green color from 395nm to 475nm, also external factors like the medium in which bacteria growth because is limited.</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ModelingTeam:BIOSINT Mexico/Modeling2013-09-28T03:41:39Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Modeling</h1> </html><br />
----<br />
----<br />
<br />
GFP tends to get fluorescents at the 395 nm because of the excitation of the protein this extend to the 475 nm where the green light is still visible, so we used this information in order to determine an equation that represents the exponential growth of the luminescent.<br />
What we pretend with this mathematic model is to represent in a numerical way, the expression of the protein in the cell according with the emission that each concentration of GFP provides. <br />
<br />
<br />
(y=a/(1+be)^(-ct))<br />
<br />
[[File:Tabla.jpg]]<br />
<br />
We used linear regression in order to get the values of the constants, using statistical methods that also are applied in the equation. The slope is the value given to b and the intersection is the value for a.<br />
<br />
<br />
y=a/(1+be^(-ct))<br />
<br />
be^(-ct)=a/y-1<br />
<br />
ln(b)-ct=ln(a/y)-ln(1)<br />
<br />
c=(ln)(b)-ln(a)-ln(y))/t<br />
<br />
<br />
[[File:Plot.jpg]]<br />
<br />
<br />
The graph represent an exponential growth of luminescent that at the same time is restricted the own GFP´s spectrum which only gives green color from 395nm to 475nm, also external factors like the medium in which bacteria growth because is limited.</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ModelingTeam:BIOSINT Mexico/Modeling2013-09-28T03:40:45Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Modeling</h1> </html><br />
----<br />
----<br />
<br />
GFP tends to get fluorescents at the 395 nm because of the excitation of the protein this extend to the 475 nm where the green light is still visible, so we used this information in order to determine an equation that represents the exponential growth of the luminescent.<br />
What we pretend with this mathematic model is to represent in a numerical way, the expression of the protein in the cell according with the emission that each concentration of GFP provides. <br />
<br />
(y=a/(1+be)^(-ct))<br />
<br />
<br />
[[File:Tabla.jpg]]<br />
<br />
We used linear regression in order to get the values of the constants, using statistical methods that also are applied in the equation. The slope is the value given to b and the intersection is the value for a.<br />
<br />
<br />
y=a/(1+be^(-ct))<br />
<br />
be^(-ct)=a/y-1<br />
<br />
ln(b)-ct=ln(a/y)-ln(1)<br />
<br />
c=(ln)(b)-ln(a)-ln(y))/t<br />
<br />
<br />
[[File:Plot.jpg]]<br />
<br />
<br />
The graph represent an exponential growth of luminescent that at the same time is restricted the own GFP´s spectrum which only gives green color from 395nm to 475nm, also external factors like the medium in which bacteria growth because is limited.</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ModelingTeam:BIOSINT Mexico/Modeling2013-09-28T03:40:19Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Modeling</h1> </html><br />
----<br />
----<br />
<br />
GFP tends to get fluorescents at the 395 nm because of the excitation of the protein this extend to the 475 nm where the green light is still visible, so we used this information in order to determine an equation that represents the exponential growth of the luminescent.<br />
What we pretend with this mathematic model is to represent in a numerical way, the expression of the protein in the cell according with the emission that each concentration of GFP provides. <br />
<br />
\\(y=a/(1+be)^(-ct))<br />
<br />
<br />
[[File:Tabla.jpg\\]]<br />
<br />
We used linear regression in order to get the values of the constants, using statistical methods that also are applied in the equation. The slope is the value given to b and the intersection is the value for a.<br />
<br />
<br />
y=a/(1+be^(-ct))<br />
<br />
be^(-ct)=a/y-1<br />
<br />
ln(b)-ct=ln(a/y)-ln(1)<br />
<br />
c=(ln)(b)-ln(a)-ln(y))/t<br />
<br />
<br />
[[File:Plot.jpg\\]]<br />
<br />
<br />
The graph represent an exponential growth of luminescent that at the same time is restricted the own GFP´s spectrum which only gives green color from 395nm to 475nm, also external factors like the medium in which bacteria growth because is limited.</div>Gabyqzhttp://2013.igem.org/File:Tabla.jpgFile:Tabla.jpg2013-09-28T03:39:55Z<p>Gabyqz: </p>
<hr />
<div></div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ModelingTeam:BIOSINT Mexico/Modeling2013-09-28T03:37:30Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Modeling</h1> </html><br />
----<br />
----<br />
<br />
GFP tends to get fluorescents at the 395 nm because of the excitation of the protein this extend to the 475 nm where the green light is still visible, so we used this information in order to determine an equation that represents the exponential growth of the luminescent.<br />
What we pretend with this mathematic model is to represent in a numerical way, the expression of the protein in the cell according with the emission that each concentration of GFP provides. <br />
(y=a/(1+be)^(-ct))<br />
<br />
X Y a= 385<br />
0 395 b= 10<br />
1 405 <br />
2 415 <br />
3 425 <br />
4 435 <br />
5 445 <br />
6 455 <br />
7 465 <br />
8 475 <br />
<br />
<br />
We used linear regression in order to get the values of the constants, using statistical methods that also are applied in the equation. The slope is the value given to b and the intersection is the value for a.<br />
<br />
<br />
y=a/(1+be^(-ct))<br />
<br />
be^(-ct)=a/y-1<br />
<br />
ln(b)-ct=ln(a/y)-ln(1)<br />
<br />
c=(ln)(b)-ln(a)-ln(y))/t<br />
<br />
<br />
[[File:Plot.jpg\\]]<br />
<br />
<br />
The graph represent an exponential growth of luminescent that at the same time is restricted the own GFP´s spectrum which only gives green color from 395nm to 475nm, also external factors like the medium in which bacteria growth because is limited.</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ModelingTeam:BIOSINT Mexico/Modeling2013-09-28T03:36:25Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Modeling</h1> </html><br />
----<br />
----<br />
<br />
GFP tends to get fluorescents at the 395 nm because of the excitation of the protein this extend to the 475 nm where the green light is still visible, so we used this information in order to determine an equation that represents the exponential growth of the luminescent.<br />
What we pretend with this mathematic model is to represent in a numerical way, the expression of the protein in the cell according with the emission that each concentration of GFP provides. <br />
(y=a/(1+be)^(-ct))<br />
<br />
X Y a= 385<br />
0 395 b= 10<br />
1 405 <br />
2 415 <br />
3 425 <br />
4 435 <br />
5 445 <br />
6 455 <br />
7 465 <br />
8 475 <br />
<br />
<br />
We used linear regression in order to get the values of the constants, using statistical methods that also are applied in the equation. The slope is the value given to b and the intersection is the value for a.<br />
<br />
<br />
y=a/(1+be^(-ct))<br />
<br />
be^(-ct)=a/y-1<br />
<br />
ln(b)-ct=ln(a/y)-ln(1)<br />
<br />
c=(ln)(b)-ln(a)-ln(y))/t<br />
<br />
<br />
[[File:Plot.jpg\\]]<br />
<br />
<br />
The graph represent an exponential growth of luminescent that at the same time is restricted the own GFP´s spectrum which only gives green color from 395nm to 475nm, also external factors like the medium in which bacteria growth because is limited.</div>Gabyqzhttp://2013.igem.org/File:Plot.jpgFile:Plot.jpg2013-09-28T03:34:46Z<p>Gabyqz: </p>
<hr />
<div></div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/Safety:_Kill_switchTeam:BIOSINT Mexico/Safety: Kill switch2013-09-28T03:27:22Z<p>Gabyqz: /* Plantaricins */</p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Safety: Kill switch</h1> </html><br />
----<br />
----<br />
<br />
=== Specific kill switch===<br />
<br />
:As an extra safety measure we adapt an specific kill switch in order than our probiotic was only capable to make the specific function for wich was design. This mechanism can be regulated through a sensor, so it will be activated only when it is desired. <br />
<br />
:Plasmid recombination occurs naturally on the microbiota, so apply a kill switch could be dangerous for the others bacterial in the intestine. For that reason we development the concept of specificity, using a method that only could act in our lactobacillus strain. <br />
<br />
So our construct could be DOUBLE SECURITY because it is strain specific and function specific. <br />
<br />
<br />
===Plantaricins===<br />
----<br />
<br />
:Bacteriocins are ribosomally synthetized antimicrobial peptides produced by bacteria.4 Unlike antibiotics, bacteriocins have a narrow killing spectrum and affect only closely related strains.7 They can be classified in different groups depending on how they are produced, their components, or the bacteria they affect.<br />
<br />
:Plantaricins are one kind of bacteriocins which belong to the IIB group. This means they are heat stable, postranslationally unmodified non-lantibiotics, and are formed by two peptides. As their name suggests, they are produced by Lactobacillus plantarum.2<br />
<br />
:Recent studies have been developed on the identification of plantaricins, their activity, and interactions among each other.<br />
<br />
:Plantaricin A (PlnA) is a peptide that has both bacteriocin and pheromone activity.5 When it is produced, Plantaricin A activates five operons which are identified as plnABCD, plnEFI, plnJKLR, plnMNOP, and plnGHSTUV. The first operon codifies for pln A itself and for several proteins involved in the transduction process. Both pln EFI and pln JKLR codify for the two peptide Plantaricins. PlnE interacts with PlnF to form PlnEF while PlnJ interacts with PlnK to form PlnJK. Research suggests that certain types of strains are more susceptible to PlnEF, and others to PlnJK.1 The other subunits of this operon codify for immunity-like proteins that protect the producer against its own plantaricins. The activity of the fourth operon is still unknown, and the fifth operon has two ORF’s, one which results in PlnGH which encodes the machinery responsible of secreting and processing the Plantaricin precursors.3<br />
<br />
:PlnEF and PlnJK have strain specific antagonist activity, which is optimum when both subunits are produced equally.1 As part of our project, we will try to create a killing mechanism which produces a plantaricin (either one or the other) without producing the immunity proteins so our Lactobacillus will be susceptible to these substances once they are outside the cell membrane. As they are strain specific, they will not affect the other members of the human microbiota, nor will they be a potential risk for human health.<br />
<br />
:Generally speaking plantaricin will be produced inside the cell and exported outside the cell through ABC transporters.6 Once outside the cell, they will adopt an amphiphilic alpha helical structure which will allow them to interact with the cell membrane and permeabilize it.5 Up until now, the only known facts are that these plantaricins create pores in the membrane and dissipate both the transmembrane electrical potential (∆Ψ) and ∆pH gradient.6<br />
<br />
[[File:ks.JPG|center|200px]]<br />
<br />
===Safety forms===<br />
<br />
----<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
REFERENCES:<br />
<br />
1Anderssen, Erlend L.; Diep, Dzung Bao; Nes, Ingolf F.; Eijsink, Vincent G. H. and Nissen-Meyer, Jon. (1998). Antagonistic Activity of Lactobacillus plantarum C11: Two New Two-Peptide Bacteriocins, Plantaricins EF and JK, and the Induction Factor Plantaricin A. [Online]. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC106311/ on April 10, 2013.<br />
<br />
2Cintas, L.M.; Casaus, M.P.; Herranz, C.; Nes, I.F. and Hernández, P.E. (2013). Review: Bacteriocins of Lactic Acid Bacteria. [Online]. Retrieved from http://fst.sagepub.com/content/7/4/281.abstract on April 10, 2013.<br />
<br />
3Diep, D. B.; Havarstein, L. S. and Nes, I.F. (1996). Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11. [Online]. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC178213/ on April 10, 2013.<br />
<br />
4Dobson, A., Cotter, P.D., Ross, R.P., Hill, C. (2012) Bacteriocin Production: a Probiotic Trait? [On-line] Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255625/ on April, 10, 2013.<br />
<br />
5Hildeng Hauge, H., Mantzilas, D., Eijsink, V.G.H., Nissen-Meyer, J. (1999) Membrane-Mimicking Entities Induce Structuring of the Two-Peptide Bacteriocins Plantaricin E/F and Plantaricin J/K. [On-line] Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC93438/ on April, 10, 2013.<br />
<br />
6Moll, Gert N.; van den Akker, Emile; Hauge, Havard H.; Nissen-Meyer, Jon; Nes, Ingolf F.; Konings, Wil N. and Driessen, Arnold J. M. (1999). Complementary and Overlapping Selectivity of the Two-Peptide Bacteriocins Plantaricin EF and JK. [Online]. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC93971/ on April 10, 2013.<br />
<br />
7Riley, M.A., Wertz,J.E. (2002) BACTERIOCINS: Evolution, Ecology, and Application. [On-line] Retrieved from http://www.bio.umass.edu/biology/riley/sites/www.bio.umass.edu.biology.riley/files/2002c%20Bacteriocins-%20Evolution,%20Ecology,%20and%20Application_002.pdf on April, 10, 2013.</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/SensorTeam:BIOSINT Mexico/Sensor2013-09-28T03:26:38Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Sensor</h1> </html><br />
----<br />
----<br />
===Xenobiotics Sensor=== <br />
<br />
:The sensor consists in the application of the model development by Debrecen in 2010, using nuclear receptors as transcriptional factors1 . The family of nuclear receptors are composed by a series of proteins which acts like sensor for lipids, steroids, hormones,endocrine-disrupting chemicals, etc.2<br />
<br />
:Several xenobiotic substances are present in the intestine and many of them result toxic for the host. One example is organochlorine pesticide, a endocrine-disrupting chemical, which is a molecule that can be found on contaminated food and represent an important toxicity source. Detection and decrease levels of pesticide could be an effective way to solve the problem .3<br />
<br />
:Nuclear receptors are ligand specific, and are composed by two basis domains: ligand binding domain (LBD) and DNA binding domain (DBD).2 In our sensor we adapt and characterized Debrecen system, which fuses specific LBD with Gal 4 DBD. 1<br />
<br />
:The system adapt TRE-CMV promoter; that has a basal expression in the absence of TetR, Gal 4; yeast transcription factor galactose 4, PXR ligand binding domain which binds organochlorine and PolyA; important for nuclear export. The complete system will be expressed in one plasmid. 1<br />
<br />
:Finally the nuclear receptor will interact with UAS, using Gal4 as specific binding region. UAS and Gal-4 forms a complex which activate the expression of the L.plantarum promoter. Development of specific nuclear receptors helps to quantify accurately the expression of the report product in relation with the ligand concentration. 4<br />
<br />
:The probiotic sensor detect organochlorine, then the nuclear receptor complex activates the promoter for the expression of linA enzyme, which catalyze the conversion of γ-hexachlorocyclohexane to 1,2,4-trichloro benzene.5Using this method our probiotic is not only available to detect organochlorine pesticides but also degrade capabilities of the molecule, contributed to decrease the presence of the toxic molecule .<br />
<br />
<br />
[[File:Detection_.jpg|center|300px]]<br />
<br />
[[File:Sensorxeno.jpg|center|400px]]<br />
<br />
<br />
===REFERENCES===<br />
<br />
----<br />
<br />
1.iGEM Debrecen (2010).Recovered from https://2010.igem.org/Team:Debrecen-Hungary/favorite<br />
<br />
2. Willson, T. et al.(2002) PXR, CAR and drug metabolism. Nature Publishing group: Volume 1<br />
<br />
3.Kojima, H, et al. (2010). Endocrine- disrupting potential of pesticides via nuclear receptors and aryl hidrocarbon receptor. Journal of health science. <br />
<br />
4. Elliot, D. & Brand A. (2002) The Gal4 system. Methods in molecular biology in Drosophila: A Fly Geneticist's Swiss Army Knife.<br />
<br />
5.Yang, J, (2011). Construction of a Genetically Engineered Microorganism that Simultaneously Degrades Organochlorine and Organophosphate Pesticides. Appl Biochem Biotechnol.</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ChassisTeam:BIOSINT Mexico/Chassis2013-09-28T03:26:00Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
__NOTOC__<br />
<br />
----<br />
<html><h1 class="myowntitle">Chassis</h1> </html><br />
----<br />
----<br />
===Lactobacillus chassis===<br />
----<br />
<br />
<br />
<br />
<br />
:This year the Biosint Mexico team will develope a smart probiotic. Throughout the competition several projects about probiotics have been present, nevertheless the main disadvantage was that most of them were not being created in a lactobacillus strain. Because of this we constructed a Lactobacillus platform for others iGEM teams. <br />
<br />
[[File:Backbonevec.jpg|center|300px]]<br />
<br />
<br />
=== L. plantarum?=== <br />
----<br />
<br />
<br />
<br />
[[File:Plantarum.jpg|right|300px]]<br />
<br />
:Lactobacillus plantarum is a gram-positive lactic acid bacterium, present naturally in dairy products, as well as in the gastrointestinal tract1. <br />
<br />
:We chose to use L. plantarum as a chassis , because of its unique ability to grow and adapt to a large number of niches2. Studies have shown that L. plantarum has the capacity to optimize its genetic material in order to adapt to different environments, especially those with high levels of carbohydrates2. Another special property of L. plantarum is that it has a high tolerance towards acids, and it is present in many fermentative processes2.<br />
<br />
:These characteristics among others make it a potential candidate for its use in different industries. Using recombinant DNA technology, this bacterium could be applied to food industries as a starter or marker in fermentative processes, in healthcare industry as a probiotic or in biofuel production2. As part of our project, we will be explore the potential of L. plantarum as a probiotic, and produce biobricks that can join all the beneficial properties of different probiotics and express it in our own chassis.<br />
<br />
===Backbones registry testing===<br />
----<br />
<br />
:Before we tried to design a backbone compatible with the biobricks registry, we make experiments in order to know if some backbones in the registry was already compatible with a lactobacillus chassis. <br />
<br />
:Tests for psB4K5, psB3K5, psB2K3, psB1A7 was made it. Unfortunatley we are not avaible to transform lacctobacillus plantarum with any of the plasmids. <br />
<br />
<br />
=== Vector design ===<br />
----<br />
<br />
[[File:backboneplant.jpg|left|200px]]<br />
<br />
<br />
:Using psb1c3 as template we design a new backbone compatible with E.coli and lactobacilli. In our case we used the bases of a staphylococcal plasmid, pUB110, that replicates by a rolling-circle mechanism via a single-stranded (ss) DNA intermediate in Bacillus subtilis and Staphylococcus aureus, and this mechanism is widespread among gram-positive bacteria . When pUB110 replicates in cells of B.subtilis or S. aureus, the plasmid regions, rep, ori, and BA3, function as a structural gene that encodes a replication protein.<br />
<br />
:The BA3 minus origin has the ability to increase stability of the plasmid in its active host (7, 33), effects of palA and the IG region on plasmid stability were examined in L. casei MSK248.. These results indicate that palA and the IG region of M13 provide increased stability to the plasmids in which they have been incorporated in L. casei.<br />
<br />
:We used the minus origin mention before and paste into psb1c3 in order to replaced the origin of replication that the vector actually have. We also add GFP to the plasmid, to measure transformation efficiency.<br />
<br />
<br />
<br />
===REFERENCES:=== <br />
----<br />
<br />
1Lee, J., Halgerson, J., Kim, J., and O’Sullivan, D. (2007). Comparative Sequence Analysis of Plasmids from Lactobacillus delbrueckii and Construction of a Shuttle Cloning Vector. [Online]. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1932812/ on April 16, 2013.<br />
<br />
2Siezen, R. and Hylckama, J. (2011). Genomic diversity and versatility of Lactobacillus plantarum, a natural metabolic engineer. [Online]. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3271238/ on April 16, 2013.</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ModelingTeam:BIOSINT Mexico/Modeling2013-09-28T03:20:11Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Modeling</h1> </html><br />
----<br />
----<br />
<br />
GFP tends to get fluorescents at the 395 nm because of the excitation of the protein this extend to the 475 nm where the green light is still visible, so we used this information in order to determine an equation that represents the exponential growth of the luminescent.<br />
What we pretend with this mathematic model is to represent in a numerical way, the expression of the protein in the cell according with the emission that each concentration of GFP provides.<br />
(y=a/(1+be)^(-ct))<br />
<br />
X Y a= 385<br />
0 395 b= 10<br />
1 405 <br />
2 415 <br />
3 425 <br />
4 435 <br />
5 445 <br />
6 455 <br />
7 465 <br />
8 475 <br />
We used linear regression in order to get the values of the constants, using statistical methods that also are applied in the equation. The slope is the value given to b and the intersection is the value for a.<br />
<br />
<br />
y=a/(1+be^(-ct))<br />
be^(-ct)=a/y-1<br />
ln(b)-ct=ln(a/y)-ln(1)<br />
c=(ln)(b)-ln(a)-ln(y))/t<br />
<br />
<br />
[[File:MSP97441g4b054f29a477d400005ab59eff9036he24.gif\\]]<br />
<br />
<br />
The graph represent an exponential growth of luminescent that at the same time is restricted the own GFP´s spectrum which only gives green color from 395nm to 475nm, also external factors like the medium in which bacteria growth because is limited.</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/PartsTeam:BIOSINT Mexico/Parts2013-09-28T03:19:38Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Parts</h1> </html><br />
----<br />
---- <br />
<br />
<groupparts>iGEM013 BIOSINT_Mexico</groupparts><br />
<br />
<br />
Lactobacillus plantarum Promoter 1: Synthetic promoter derived from rRNA library promoters extracted from the L. plantarum WCFS1 genome. Cloned and expression in E.coli, measured activity with GFP ligation.<br />
<br />
Lactobacillus plantarum Promoter 2: Synthetic promoter derived from rRNA library promoters extracted from the L. plantarum WCFS1 genome. Cloned and expression in E.coli, measured activity with GFP ligation.<br />
<br />
RBS for Lactobacillus plantarum: Ribosome binding site design for L. plantarum</div>Gabyqzhttp://2013.igem.org/File:MSP97441g4b054f29a477d400005ab59eff9036he24.gifFile:MSP97441g4b054f29a477d400005ab59eff9036he24.gif2013-09-28T03:18:53Z<p>Gabyqz: </p>
<hr />
<div></div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/PartsTeam:BIOSINT Mexico/Parts2013-09-28T03:18:23Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Parts</h1> </html><br />
----<br />
----<br />
<br />
<br />
Lactobacillus plantarum Promoter 1: Synthetic promoter derived from rRNA library promoters extracted from the L. plantarum WCFS1 genome. Cloned and expression in E.coli ligated with GFP.<br />
<br />
Lactobacillus plantarum Promoter 2: Synthetic promoter derived from rRNA library promoters extracted from the L. plantarum WCFS1 genome. Cloned and expression in E.coli ligated with GFP. <br />
<br />
RBS for Lactobacillus plantarum: Ribosome binding site design for L. plantarum <br />
<br />
<br />
<groupparts>iGEM013 BIOSINT_Mexico</groupparts></div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ModelingTeam:BIOSINT Mexico/Modeling2013-09-28T03:17:14Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Modeling</h1> </html><br />
----<br />
----<br />
<br />
GFP tends to get fluorescents at the 395 nm because of the excitation of the protein this extend to the 475 nm where the green light is still visible, so we used this information in order to determine an equation that represents the exponential growth of the luminescent.<br />
What we pretend with this mathematic model is to represent in a numerical way, the expression of the protein in the cell according with the emission that each concentration of GFP provides.<br />
〖y=a/(1+be〗^(-ct))<br />
<br />
X Y a= 385<br />
0 395 b= 10<br />
1 405 <br />
2 415 <br />
3 425 <br />
4 435 <br />
5 445 <br />
6 455 <br />
7 465 <br />
8 475 <br />
We used linear regression in order to get the values of the constants, using statistical methods that also are applied in the equation. The slope is the value given to b and the intersection is the value for a.<br />
<br />
<br />
y=a/(1+be^(-ct) )<br />
be^(-ct)=a/y-1<br />
ln(b)-ct=ln(a/y)-ln(1)<br />
c=〖(ln〗(b)-ln(a)-ln(y))/t<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
The graph represent an exponential growth of luminescent that at the same time is restricted the own GFP´s spectrum which only gives green color from 395nm to 475nm, also external factors like the medium in which bacteria growth because is limited.</div>Gabyqzhttp://2013.igem.org/File:Fac1.jpgFile:Fac1.jpg2013-09-28T03:08:36Z<p>Gabyqz: </p>
<hr />
<div></div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/SponsorsTeam:BIOSINT Mexico/Sponsors2013-09-28T03:07:16Z<p>Gabyqz: Created page with "{{:Team:BIOSINT_Mexico/Header}} __NOTOC__ ---- <html><h1 class="myowntitle">Sponsors</h1> </html> ---- ----"</p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
__NOTOC__<br />
<br />
----<br />
<html><h1 class="myowntitle">Sponsors</h1> </html><br />
----<br />
----</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/HeaderTeam:BIOSINT Mexico/Header2013-09-28T03:06:51Z<p>Gabyqz: </p>
<hr />
<div><html><br />
<style><br />
<br />
body{<br />
background: #F2F0F0;<br />
<br />
<br />
}<br />
#p-logo{<br />
display:none;<br />
<br />
<br />
}<br />
#top-section {<br />
height:130px ! important;<br />
border: 0;<br />
width:100%;<br />
background: #bfd255; /* Old browsers */<br />
background: -moz-linear-gradient(top, #bfd255 0%, #8eb92a 50%, #72aa00 51%, #9ecb2d 100%); /* FF3.6+ */<br />
background: -webkit-gradient(linear, left top, left bottom, color-stop(0%,#bfd255), color-stop(50%,#8eb92a), color-stop(51%,#72aa00), color-stop(100%,#9ecb2d)); /* Chrome,Safari4+ */<br />
background: -webkit-linear-gradient(top, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* Chrome10+,Safari5.1+ */<br />
background: -o-linear-gradient(top, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* Opera 11.10+ */<br />
background: -ms-linear-gradient(top, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* IE10+ */<br />
background: linear-gradient(to bottom, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* W3C */<br />
filter: progid:DXImageTransform.Microsoft.gradient( startColorstr='#bfd255', endColorstr='#9ecb2d',GradientType=0 ); /* IE6-9 */<br />
}<br />
<br />
#menubar{<br />
margin-left: 175px;<br />
}<br />
.right-menu li a {<br />
background-color: transparent;<br />
}<br />
#searchInput {<br />
float:right;<br />
height: 25px;<br />
width: 170px;<br />
font-size: 14px;<br />
border: 1px solid #000000;<br />
border-radius: 5px 5px 5px 5px;<br />
-webkit-border-radius: 5px 5px 5px 5px;<br />
-moz-border-radius: 5px 5px 5px 5px;<br />
box-shadow:inset 1px 1px 3px #000;<br />
-webkit-box-shadow:inset 1px 1px 3px #000;<br />
-moz-box-shadow:inset 1px 1px 3px #000;<br />
padding-left:5px;<br />
}<br />
input.searchButton{<br />
color: #ffffff;<br />
}<br />
input.searchButton:hover{<br />
color: #4682b4;<br />
}<br />
<br />
#footer-box a{<br />
color:#fff;<br />
}<br />
#content {<br />
background: #fff;<br />
border: 0;<br />
color: #333;<br />
margin: 50 auto;<br />
<br />
<br />
position: relative;<br />
width: 85%;<br />
z-index: 2;<br />
border-radius: 5px 5px;<br />
text-align: justify;<br />
<br />
box-shadow: 0px 0px 20px #333;<br />
-webkit-box-shadow: 0px 0px 20px #333;<br />
-moz-box-shadow: 0px 0px 20px #333;<br />
}<br />
#content img{<br />
margin: 10px;<br />
box-shadow: 0px 0px 0px #333;<br />
-webkit-box-shadow: 0px 0px 0px #333;<br />
-moz-box-shadow: 0px 0px 0px #333;<br />
}<br />
#bodyContent{<br />
width:95%;<br />
background:#fff;<br />
margin:0 auto;<br />
<br />
}<br />
<br />
.firstHeading {<br />
/*text-shadow: 1px 1px 2px #000;<br />
margin-left: 20px;<br />
color:#ff0000;<br />
font-size: 36px;<br />
font-weight: bold;<br />
font-family: Helvetica,Arial,sans-serif;*/<br />
display:none;<br />
}<br />
.myowntitle{<br />
text-shadow: 1px 1px 2px #000;<br />
margin-left: 30px;<br />
color:#008000;<br />
font-size: 36px;<br />
font-weight: bold;<br />
font-family: "Comic Sans MS", cursive, sans-serif;<br />
}<br />
h1{<br />
border-bottom:0;<br />
}<br />
<br />
#catlinks {<br />
background-color: transparent;<br />
border: 0;<br />
clear: both;<br />
margin:10px 0 ;<br />
padding: 10px;<br />
}<br />
#my_own_top{<br />
margin: 0 auto;<br />
position: absolute;<br />
top:-186px;<br />
width: 975px;<br />
height: 130px;<br />
overflow:hidden;<br />
}<br />
#my_own_top h1{<br />
<br />
color:#fff;<br />
font-size:50px;<br />
font-weight:bold;<br />
padding:0;<br />
height:50px;<br />
width:300px;<br />
margin:100px 30px 0 350px;<br />
padding-left:30px;<br />
position:relative;<br />
}<br />
#my_own_top img.logo{<br />
float:right;<br />
position:relative;<br />
}<br />
#my_own_top img{<br />
position:relative;<br />
}<br />
#navigation{<br />
float:left;<br />
width:100%;<br />
height: 40px;<br />
background:#000;<br />
margin-top:-20px;<br />
border-bottom-right-radius:8px;<br />
border-bottom-left-radius:8px;<br />
}<br />
#navigation ul{<br />
list-style-type: none !important;<br />
list-style-image: none;<br />
}<br />
#navigation ul li{<br />
width:130px;<br />
min-height: 35px;<br />
text-align:center;<br />
vertical-align: middle;<br />
position: relative;<br />
float: left;<br />
margin-right: 10px;<br />
background:#000;<br />
}<br />
#navigation a{<br />
text-decoration:none;<br />
font-size: 17px;<br />
color: #FFF;<br />
text-shadow: 1px 1px 5px #fff;<br />
}<br />
#navigation a:hover{<br />
color:#fff;<br />
background:#2E2E2E;<br />
font-size:1.4em;<br />
text-shadow:1px 1px 5px #81F7F3;<br />
}<br />
<br />
#navigation li:hover{<br />
background:#2E2E2E;<br />
<br />
}<br />
.menu_body {display:none; width:184px;margin-left: 0px; margin-top: 10px; z-index:999;}<br />
.menu_body li{background:#fffafa; background-image: none !important;z-index:999; margin:0; border-radius: 0px;}<br />
.menu_body li.alt{background:#fffafa;background-image: none; border-radius: 0px;}<br />
.menu_body li a{color:#fffafa; text-decoration:none; padding:10px; display:block;}<br />
.menu_body li a:hover{ font-weight:bold;}<br />
p{ font-style:italic; padding-left:20px;padding:right:20px; margin-right:20px;}<br />
<br />
<br />
<br />
</style><br />
<br />
<div id="my_own_top"><br />
<a href="https://2011.igem.org/Team:ITESM_Mexico" ><img src="http://s22.postimg.org/7n99nq2b5/A_lo_largo.png" alt="Biobrickoatl Home" class="logo" /></a><br />
<div style="float:center;position:absolute;" ><br />
<h1>SmartPro</h1><br />
<h1 style="font-size: 36px; color:#db0976; height:50px;">Smartpro</h1><br />
</div><br />
<a href="https://igem.org" ><img src="http://deviatan.com/sponsoring/igem_light.png" alt="Igem" style="margin:50px 100px 50px 0;"/></a><br />
<a href="http://postimg.org/image/6ptdqlbp5/" ><img src="http://s12.postimg.org/6ptdqlbp5/Logotipo_Definitivo.png" alt="LogotipoDefinitivo" style="margin:0px 0px 42px 600;" /></a><br />
<br />
<br />
</div><br />
<br />
<div id="navigation"><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico">Home</a><br />
<br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Team">Team</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Who we are">Who we are</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Acknowledgment">Special Thanks</a></li><br />
<li><a href="https://igem.org/Team.cgi">Official team page</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Sponsors">Sponsors</a></li><br />
</ul><br />
<br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Project">Project</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Abstract">Abstract</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Chassis">Chassis</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Sensor">Sensor</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Safety: Kill switch">Safety: Kill switch</a><br />
</li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Construction">Construction</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Results">Results</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Future research">Future research</a></li><br />
<br />
</ul><br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Parts">Parts</a><br />
<br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Modeling">Modeling</a><br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Human Practice">Human Practice</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Probiotics: iGEM contribution">Probiotics: iGEM contribution</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Workshop">Workshop</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Publicity">Publicity</a></li> <br />
<br />
</ul><br />
</li> <br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Notebook">Notebook</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Protocols">Protocols</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOINT_Mexico/Lab work">Lab Work</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOINT_Mexico/Gallery">Gallery</a></li><br />
</ul><br />
</li><br />
<br />
</ul><br />
</div><br />
<script type="text/javascript" src="https://ajax.googleapis.com/ajax/libs/jquery/1.6.2/jquery.min.js"></script><br />
<br />
<script type="text/javascript"><br />
$(document).ready(function () {<br />
<br />
<br />
<br />
$("ul.menu_body li:even").addClass("alt");<br />
$('div#navigation ul li a').mouseover(function () {<br />
//alert(this);<br />
$(this).parent().find("ul.menu_body").slideDown('medium');<br />
$(this).parent().hover(function() {<br />
}, function(){<br />
$(this).parent().find("ul.menu_body").slideUp('medium');<br />
});<br />
});<br />
<br />
$('ul.menu_body li a').mouseover(function () {<br />
$(this).animate({ fontSize: "16px" }, 50 );<br />
});<br />
$('ul.menu_body li a').mouseout(function () {<br />
$(this).animate({ fontSize: "15px" }, 50 );<br />
});<br />
<br />
<br />
<br />
<br />
<br />
});<br />
<br />
</script><br />
<br />
</html></div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/HeaderTeam:BIOSINT Mexico/Header2013-09-28T03:05:46Z<p>Gabyqz: </p>
<hr />
<div><html><br />
<style><br />
<br />
body{<br />
background: #F2F0F0;<br />
<br />
<br />
}<br />
#p-logo{<br />
display:none;<br />
<br />
<br />
}<br />
#top-section {<br />
height:130px ! important;<br />
border: 0;<br />
width:100%;<br />
background: #bfd255; /* Old browsers */<br />
background: -moz-linear-gradient(top, #bfd255 0%, #8eb92a 50%, #72aa00 51%, #9ecb2d 100%); /* FF3.6+ */<br />
background: -webkit-gradient(linear, left top, left bottom, color-stop(0%,#bfd255), color-stop(50%,#8eb92a), color-stop(51%,#72aa00), color-stop(100%,#9ecb2d)); /* Chrome,Safari4+ */<br />
background: -webkit-linear-gradient(top, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* Chrome10+,Safari5.1+ */<br />
background: -o-linear-gradient(top, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* Opera 11.10+ */<br />
background: -ms-linear-gradient(top, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* IE10+ */<br />
background: linear-gradient(to bottom, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* W3C */<br />
filter: progid:DXImageTransform.Microsoft.gradient( startColorstr='#bfd255', endColorstr='#9ecb2d',GradientType=0 ); /* IE6-9 */<br />
}<br />
<br />
#menubar{<br />
margin-left: 175px;<br />
}<br />
.right-menu li a {<br />
background-color: transparent;<br />
}<br />
#searchInput {<br />
float:right;<br />
height: 25px;<br />
width: 170px;<br />
font-size: 14px;<br />
border: 1px solid #000000;<br />
border-radius: 5px 5px 5px 5px;<br />
-webkit-border-radius: 5px 5px 5px 5px;<br />
-moz-border-radius: 5px 5px 5px 5px;<br />
box-shadow:inset 1px 1px 3px #000;<br />
-webkit-box-shadow:inset 1px 1px 3px #000;<br />
-moz-box-shadow:inset 1px 1px 3px #000;<br />
padding-left:5px;<br />
}<br />
input.searchButton{<br />
color: #ffffff;<br />
}<br />
input.searchButton:hover{<br />
color: #4682b4;<br />
}<br />
<br />
#footer-box a{<br />
color:#fff;<br />
}<br />
#content {<br />
background: #fff;<br />
border: 0;<br />
color: #333;<br />
margin: 50 auto;<br />
<br />
<br />
position: relative;<br />
width: 85%;<br />
z-index: 2;<br />
border-radius: 5px 5px;<br />
text-align: justify;<br />
<br />
box-shadow: 0px 0px 20px #333;<br />
-webkit-box-shadow: 0px 0px 20px #333;<br />
-moz-box-shadow: 0px 0px 20px #333;<br />
}<br />
#content img{<br />
margin: 10px;<br />
box-shadow: 0px 0px 0px #333;<br />
-webkit-box-shadow: 0px 0px 0px #333;<br />
-moz-box-shadow: 0px 0px 0px #333;<br />
}<br />
#bodyContent{<br />
width:95%;<br />
background:#fff;<br />
margin:0 auto;<br />
<br />
}<br />
<br />
.firstHeading {<br />
/*text-shadow: 1px 1px 2px #000;<br />
margin-left: 20px;<br />
color:#ff0000;<br />
font-size: 36px;<br />
font-weight: bold;<br />
font-family: Helvetica,Arial,sans-serif;*/<br />
display:none;<br />
}<br />
.myowntitle{<br />
text-shadow: 1px 1px 2px #000;<br />
margin-left: 30px;<br />
color:#008000;<br />
font-size: 36px;<br />
font-weight: bold;<br />
font-family: "Comic Sans MS", cursive, sans-serif;<br />
}<br />
h1{<br />
border-bottom:0;<br />
}<br />
<br />
#catlinks {<br />
background-color: transparent;<br />
border: 0;<br />
clear: both;<br />
margin:10px 0 ;<br />
padding: 10px;<br />
}<br />
#my_own_top{<br />
margin: 0 auto;<br />
position: absolute;<br />
top:-186px;<br />
width: 975px;<br />
height: 130px;<br />
overflow:hidden;<br />
}<br />
#my_own_top h1{<br />
<br />
color:#fff;<br />
font-size:50px;<br />
font-weight:bold;<br />
padding:0;<br />
height:50px;<br />
width:300px;<br />
margin:100px 30px 0 350px;<br />
padding-left:30px;<br />
position:relative;<br />
}<br />
#my_own_top img.logo{<br />
float:right;<br />
position:relative;<br />
}<br />
#my_own_top img{<br />
position:relative;<br />
}<br />
#navigation{<br />
float:left;<br />
width:100%;<br />
height: 40px;<br />
background:#000;<br />
margin-top:-20px;<br />
border-bottom-right-radius:8px;<br />
border-bottom-left-radius:8px;<br />
}<br />
#navigation ul{<br />
list-style-type: none !important;<br />
list-style-image: none;<br />
}<br />
#navigation ul li{<br />
width:130px;<br />
min-height: 35px;<br />
text-align:center;<br />
vertical-align: middle;<br />
position: relative;<br />
float: left;<br />
margin-right: 10px;<br />
background:#000;<br />
}<br />
#navigation a{<br />
text-decoration:none;<br />
font-size: 17px;<br />
color: #FFF;<br />
text-shadow: 1px 1px 5px #fff;<br />
}<br />
#navigation a:hover{<br />
color:#fff;<br />
background:#2E2E2E;<br />
font-size:1.4em;<br />
text-shadow:1px 1px 5px #81F7F3;<br />
}<br />
<br />
#navigation li:hover{<br />
background:#2E2E2E;<br />
<br />
}<br />
.menu_body {display:none; width:184px;margin-left: 0px; margin-top: 10px; z-index:999;}<br />
.menu_body li{background:#fffafa; background-image: none !important;z-index:999; margin:0; border-radius: 0px;}<br />
.menu_body li.alt{background:#fffafa;background-image: none; border-radius: 0px;}<br />
.menu_body li a{color:#fffafa; text-decoration:none; padding:10px; display:block;}<br />
.menu_body li a:hover{ font-weight:bold;}<br />
p{ font-style:italic; padding-left:20px;padding:right:20px; margin-right:20px;}<br />
<br />
<br />
<br />
</style><br />
<br />
<div id="my_own_top"><br />
<a href="https://2011.igem.org/Team:ITESM_Mexico" ><img src="http://s22.postimg.org/7n99nq2b5/A_lo_largo.png" alt="Biobrickoatl Home" class="logo" /></a><br />
<div style="float:center;position:absolute;" ><br />
<h1>SmartPro</h1><br />
<h1 style="font-size: 36px; color:#db0976; height:50px;">Smartpro</h1><br />
</div><br />
<a href="https://igem.org" ><img src="http://deviatan.com/sponsoring/igem_light.png" alt="Igem" style="margin:50px 100px 50px 0;"/></a><br />
<a href="http://postimg.org/image/6ptdqlbp5/" ><img src="http://s12.postimg.org/6ptdqlbp5/Logotipo_Definitivo.png" alt="LogotipoDefinitivo" style="margin:0px 0px 42px 600;" /></a><br />
<br />
<br />
</div><br />
<br />
<div id="navigation"><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico">Home</a><br />
<br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Team">Team</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Who we are">Who we are</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Acknowledgment">Special Thanks</a></li><br />
<li><a href="https://igem.org/Team.cgi">Official team page</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico">Sponsors</a></li><br />
</ul><br />
<br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Project">Project</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Abstract">Abstract</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Chassis">Chassis</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Sensor">Sensor</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Safety: Kill switch">Safety: Kill switch</a><br />
</li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Construction">Construction</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Results">Results</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Future research">Future research</a></li><br />
<br />
</ul><br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Parts">Parts</a><br />
<br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Modeling">Modeling</a><br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Human Practice">Human Practice</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Probiotics: iGEM contribution">Probiotics: iGEM contribution</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Workshop">Workshop</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Publicity">Publicity</a></li> <br />
<br />
</ul><br />
</li> <br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Notebook">Notebook</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Protocols">Protocols</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOINT_Mexico/Lab work">Lab Work</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOINT_Mexico/Gallery">Gallery</a></li><br />
</ul><br />
</li><br />
<br />
</ul><br />
</div><br />
<script type="text/javascript" src="https://ajax.googleapis.com/ajax/libs/jquery/1.6.2/jquery.min.js"></script><br />
<br />
<script type="text/javascript"><br />
$(document).ready(function () {<br />
<br />
<br />
<br />
$("ul.menu_body li:even").addClass("alt");<br />
$('div#navigation ul li a').mouseover(function () {<br />
//alert(this);<br />
$(this).parent().find("ul.menu_body").slideDown('medium');<br />
$(this).parent().hover(function() {<br />
}, function(){<br />
$(this).parent().find("ul.menu_body").slideUp('medium');<br />
});<br />
});<br />
<br />
$('ul.menu_body li a').mouseover(function () {<br />
$(this).animate({ fontSize: "16px" }, 50 );<br />
});<br />
$('ul.menu_body li a').mouseout(function () {<br />
$(this).animate({ fontSize: "15px" }, 50 );<br />
});<br />
<br />
<br />
<br />
<br />
<br />
});<br />
<br />
</script><br />
<br />
</html></div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/HeaderTeam:BIOSINT Mexico/Header2013-09-28T02:59:57Z<p>Gabyqz: </p>
<hr />
<div><html><br />
<style><br />
<br />
body{<br />
background: #F2F0F0;<br />
<br />
<br />
}<br />
#p-logo{<br />
display:none;<br />
<br />
<br />
}<br />
#top-section {<br />
height:130px ! important;<br />
border: 0;<br />
width:100%;<br />
background: #bfd255; /* Old browsers */<br />
background: -moz-linear-gradient(top, #bfd255 0%, #8eb92a 50%, #72aa00 51%, #9ecb2d 100%); /* FF3.6+ */<br />
background: -webkit-gradient(linear, left top, left bottom, color-stop(0%,#bfd255), color-stop(50%,#8eb92a), color-stop(51%,#72aa00), color-stop(100%,#9ecb2d)); /* Chrome,Safari4+ */<br />
background: -webkit-linear-gradient(top, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* Chrome10+,Safari5.1+ */<br />
background: -o-linear-gradient(top, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* Opera 11.10+ */<br />
background: -ms-linear-gradient(top, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* IE10+ */<br />
background: linear-gradient(to bottom, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* W3C */<br />
filter: progid:DXImageTransform.Microsoft.gradient( startColorstr='#bfd255', endColorstr='#9ecb2d',GradientType=0 ); /* IE6-9 */<br />
}<br />
<br />
#menubar{<br />
margin-left: 175px;<br />
}<br />
.right-menu li a {<br />
background-color: transparent;<br />
}<br />
#searchInput {<br />
float:right;<br />
height: 25px;<br />
width: 170px;<br />
font-size: 14px;<br />
border: 1px solid #000000;<br />
border-radius: 5px 5px 5px 5px;<br />
-webkit-border-radius: 5px 5px 5px 5px;<br />
-moz-border-radius: 5px 5px 5px 5px;<br />
box-shadow:inset 1px 1px 3px #000;<br />
-webkit-box-shadow:inset 1px 1px 3px #000;<br />
-moz-box-shadow:inset 1px 1px 3px #000;<br />
padding-left:5px;<br />
}<br />
input.searchButton{<br />
color: #ffffff;<br />
}<br />
input.searchButton:hover{<br />
color: #4682b4;<br />
}<br />
<br />
#footer-box a{<br />
color:#fff;<br />
}<br />
#content {<br />
background: #fff;<br />
border: 0;<br />
color: #333;<br />
margin: 50 auto;<br />
<br />
<br />
position: relative;<br />
width: 85%;<br />
z-index: 2;<br />
border-radius: 5px 5px;<br />
text-align: justify;<br />
<br />
box-shadow: 0px 0px 20px #333;<br />
-webkit-box-shadow: 0px 0px 20px #333;<br />
-moz-box-shadow: 0px 0px 20px #333;<br />
}<br />
#content img{<br />
margin: 10px;<br />
box-shadow: 0px 0px 0px #333;<br />
-webkit-box-shadow: 0px 0px 0px #333;<br />
-moz-box-shadow: 0px 0px 0px #333;<br />
}<br />
#bodyContent{<br />
width:95%;<br />
background:#fff;<br />
margin:0 auto;<br />
<br />
}<br />
<br />
.firstHeading {<br />
/*text-shadow: 1px 1px 2px #000;<br />
margin-left: 20px;<br />
color:#ff0000;<br />
font-size: 36px;<br />
font-weight: bold;<br />
font-family: Helvetica,Arial,sans-serif;*/<br />
display:none;<br />
}<br />
.myowntitle{<br />
text-shadow: 1px 1px 2px #000;<br />
margin-left: 30px;<br />
color:#008000;<br />
font-size: 36px;<br />
font-weight: bold;<br />
font-family: "Comic Sans MS", cursive, sans-serif;<br />
}<br />
h1{<br />
border-bottom:0;<br />
}<br />
<br />
#catlinks {<br />
background-color: transparent;<br />
border: 0;<br />
clear: both;<br />
margin:10px 0 ;<br />
padding: 10px;<br />
}<br />
#my_own_top{<br />
margin: 0 auto;<br />
position: absolute;<br />
top:-186px;<br />
width: 975px;<br />
height: 130px;<br />
overflow:hidden;<br />
}<br />
#my_own_top h1{<br />
<br />
color:#fff;<br />
font-size:50px;<br />
font-weight:bold;<br />
padding:0;<br />
height:50px;<br />
width:300px;<br />
margin:100px 30px 0 350px;<br />
padding-left:30px;<br />
position:relative;<br />
}<br />
#my_own_top img.logo{<br />
float:right;<br />
position:relative;<br />
}<br />
#my_own_top img{<br />
position:relative;<br />
}<br />
#navigation{<br />
float:left;<br />
width:100%;<br />
height: 40px;<br />
background:#000;<br />
margin-top:-20px;<br />
border-bottom-right-radius:8px;<br />
border-bottom-left-radius:8px;<br />
}<br />
#navigation ul{<br />
list-style-type: none !important;<br />
list-style-image: none;<br />
}<br />
#navigation ul li{<br />
width:130px;<br />
min-height: 35px;<br />
text-align:center;<br />
vertical-align: middle;<br />
position: relative;<br />
float: left;<br />
margin-right: 10px;<br />
background:#000;<br />
}<br />
#navigation a{<br />
text-decoration:none;<br />
font-size: 17px;<br />
color: #FFF;<br />
text-shadow: 1px 1px 5px #fff;<br />
}<br />
#navigation a:hover{<br />
color:#fff;<br />
background:#2E2E2E;<br />
font-size:1.4em;<br />
text-shadow:1px 1px 5px #81F7F3;<br />
}<br />
<br />
#navigation li:hover{<br />
background:#2E2E2E;<br />
<br />
}<br />
.menu_body {display:none; width:184px;margin-left: 0px; margin-top: 10px; z-index:999;}<br />
.menu_body li{background:#fffafa; background-image: none !important;z-index:999; margin:0; border-radius: 0px;}<br />
.menu_body li.alt{background:#fffafa;background-image: none; border-radius: 0px;}<br />
.menu_body li a{color:#fffafa; text-decoration:none; padding:10px; display:block;}<br />
.menu_body li a:hover{ font-weight:bold;}<br />
p{ font-style:italic; padding-left:20px;padding:right:20px; margin-right:20px;}<br />
<br />
<br />
<br />
</style><br />
<br />
<div id="my_own_top"><br />
<a href="https://2011.igem.org/Team:ITESM_Mexico" ><img src="http://s22.postimg.org/7n99nq2b5/A_lo_largo.png" alt="Biobrickoatl Home" class="logo" /></a><br />
<div style="float:center;position:absolute;" ><br />
<h1>SmartPro</h1><br />
<h1 style="font-size: 36px; color:#db0976; height:50px;">Smartpro</h1><br />
</div><br />
<a href="https://igem.org" ><img src="http://deviatan.com/sponsoring/igem_light.png" alt="Igem" style="margin:50px 100px 50px 0;"/></a><br />
<a href="http://postimg.org/image/6ptdqlbp5/" ><img src="http://s12.postimg.org/6ptdqlbp5/Logotipo_Definitivo.png" alt="LogotipoDefinitivo" style="margin:0px 0px 42px 600;" /></a><br />
<br />
<br />
</div><br />
<br />
<div id="navigation"><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico">Home</a><br />
<br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Team">Team</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Who we are">Who we are</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Acknowledgment">Special Thanks</a></li><br />
<li><a href="https://igem.org/Team.cgi">Official team page</a></li><br />
<li><a href="https://igem.org/Team:BIOSINT_Mexico">Sponsors</a></li><br />
</ul><br />
<br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Project">Project</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Abstract">Abstract</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Chassis">Chassis</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Sensor">Sensor</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Safety: Kill switch">Safety: Kill switch</a><br />
</li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Construction">Construction</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Results">Results</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Future research">Future research</a></li><br />
<br />
</ul><br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Parts">Parts</a><br />
<br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Modeling">Modeling</a><br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Human Practice">Human Practice</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Probiotics: iGEM contribution">Probiotics: iGEM contribution</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Workshop">Workshop</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Publicity">Publicity</a></li> <br />
<br />
</ul><br />
</li> <br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Notebook">Notebook</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Protocols">Protocols</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOINT_Mexico/Lab work">Lab Work</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOINT_Mexico/Gallery">Gallery</a></li><br />
</ul><br />
</li><br />
<br />
</ul><br />
</div><br />
<script type="text/javascript" src="https://ajax.googleapis.com/ajax/libs/jquery/1.6.2/jquery.min.js"></script><br />
<br />
<script type="text/javascript"><br />
$(document).ready(function () {<br />
<br />
<br />
<br />
$("ul.menu_body li:even").addClass("alt");<br />
$('div#navigation ul li a').mouseover(function () {<br />
//alert(this);<br />
$(this).parent().find("ul.menu_body").slideDown('medium');<br />
$(this).parent().hover(function() {<br />
}, function(){<br />
$(this).parent().find("ul.menu_body").slideUp('medium');<br />
});<br />
});<br />
<br />
$('ul.menu_body li a').mouseover(function () {<br />
$(this).animate({ fontSize: "16px" }, 50 );<br />
});<br />
$('ul.menu_body li a').mouseout(function () {<br />
$(this).animate({ fontSize: "15px" }, 50 );<br />
});<br />
<br />
<br />
<br />
<br />
<br />
});<br />
<br />
</script><br />
<br />
</html></div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ResultsTeam:BIOSINT Mexico/Results2013-09-28T02:57:23Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Results</h1> </html><br />
----<br />
----<br />
<br />
[[File:Piezasgens.jpg|right|300px]]<br />
<br />
Plasmid extraction from synthetized parts <br />
<br />
pUC57: 2710 bp<br />
<br />
*In the lane 1 is the ladder of 5000 bp<br />
<br />
*The sample of the lane 3 is the promoter 1, inserted sequence: 103 bp<br />
<br />
*The sample of the lane 5 is the promoter 2, inserted sequence: 103 bp<br />
<br />
*The sample of the lane 7 is the RBS, inserted sequence: 76 bp<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
<br />
<br />
[[File:Piezas_PsB1C3.jpg|right|300px]]<br />
<br />
<br />
<br />
<br />
<br />
Parts clonning in pSB1C3: 2070 bp<br />
<br />
<br />
*In the lane 1 is the ladder of 3000 bp<br />
<br />
*The sample of the lane 3 is the promoter 2<br />
<br />
*The sample of the lane 4 is the promoter 1<br />
<br />
*The sample of the lane 7 is the RBS<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
<br />
[[File:Plasmid_GFP.jpg|right|300px]]<br />
<br />
<br />
<br />
<br />
<br />
Ligation Promoters with GFP<br />
<br />
<br />
*In the lane 1 is the ladder of 5000 bp<br />
<br />
*The sample of the lane 3 is the Plasmid GFP<br />
<br />
*The sample of the lane 5 is the Plasmid GFP<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[File:P1_expression.jpg|center|300px]]<br />
<br />
GFP expression for L.Plantarum promoter cloned in E.coli<br />
<br />
----<br />
<br />
[[File:P2_expression.jpg|center|300px]]<br />
<br />
GFP expression for L.Plantarum promoter 2 cloned in E.coli</div>Gabyqzhttp://2013.igem.org/File:P2_expression.jpgFile:P2 expression.jpg2013-09-28T02:54:21Z<p>Gabyqz: </p>
<hr />
<div></div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ResultsTeam:BIOSINT Mexico/Results2013-09-28T02:52:51Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Results</h1> </html><br />
----<br />
----<br />
<br />
[[File:Piezasgens.jpg|right|300px]]<br />
<br />
Plasmid extraction from synthetized parts <br />
<br />
pUC57: 2710 bp<br />
<br />
*In the lane 1 is the ladder of 5000 bp<br />
<br />
*The sample of the lane 3 is the promoter 1, inserted sequence: 103 bp<br />
<br />
*The sample of the lane 5 is the promoter 2, inserted sequence: 103 bp<br />
<br />
*The sample of the lane 7 is the RBS, inserted sequence: 76 bp<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
<br />
<br />
[[File:Piezas_PsB1C3.jpg|right|300px]]<br />
<br />
<br />
<br />
<br />
<br />
Parts clonning in pSB1C3: 2070 bp<br />
<br />
<br />
*In the lane 1 is the ladder of 3000 bp<br />
<br />
*The sample of the lane 3 is the promoter 2<br />
<br />
*The sample of the lane 4 is the promoter 1<br />
<br />
*The sample of the lane 7 is the RBS<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
<br />
[[File:Plasmid_GFP.jpg|right|300px]]<br />
<br />
<br />
<br />
<br />
<br />
Ligation Promoters with GFP<br />
<br />
<br />
*In the lane 1 is the ladder of 5000 bp<br />
<br />
*The sample of the lane 3 is the Plasmid GFP<br />
<br />
*The sample of the lane 5 is the Plasmid GFP<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[File:P1_expression.jpg|center|300px]]<br />
<br />
<br />
GFP expression for L.Plantarum promoter cloned in E.coli</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ResultsTeam:BIOSINT Mexico/Results2013-09-28T02:52:03Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Results</h1> </html><br />
----<br />
----<br />
<br />
[[File:Piezasgens.jpg|right|300px]]<br />
<br />
Plasmid extraction from synthetized parts <br />
<br />
pUC57: 2710 bp<br />
<br />
*In the lane 1 is the ladder of 5000 bp<br />
<br />
*The sample of the lane 3 is the promoter 1, inserted sequence: 103 bp<br />
<br />
*The sample of the lane 5 is the promoter 2, inserted sequence: 103 bp<br />
<br />
*The sample of the lane 7 is the RBS, inserted sequence: 76 bp<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
<br />
<br />
[[File:Piezas_PsB1C3.jpg|right|300px]]<br />
<br />
<br />
<br />
<br />
<br />
Parts clonning in pSB1C3: 2070 bp<br />
<br />
<br />
*In the lane 1 is the ladder of 3000 bp<br />
<br />
*The sample of the lane 3 is the promoter 2<br />
<br />
*The sample of the lane 4 is the promoter 1<br />
<br />
*The sample of the lane 7 is the RBS<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
<br />
[[File:Plasmid_GFP.jpg|right|300px]]<br />
<br />
<br />
<br />
<br />
<br />
Ligation Promoters with GFP<br />
<br />
<br />
*In the lane 1 is the ladder of 5000 bp<br />
<br />
*The sample of the lane 3 is the Plasmid GFP<br />
<br />
*The sample of the lane 5 is the Plasmid GFP<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[File:P1_expression.jpg|center|300px]]<br />
<br />
<br />
GFP expression for L.Plantarum promoter cloned in E.coli</div>Gabyqzhttp://2013.igem.org/File:P1_expression.jpgFile:P1 expression.jpg2013-09-28T02:48:48Z<p>Gabyqz: </p>
<hr />
<div></div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/AcknowledgmentTeam:BIOSINT Mexico/Acknowledgment2013-09-28T02:43:07Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Special Thanks</h1> </html><br />
----<br />
----<br />
<br />
<br />
:We would like to give our most significative thanks to all the people that supported and helped us. Specially thanks to the people that work in our university, '''Instituto Tecnologico de Estudios Superiores de Monterrey Campus Queretaro''', because of them we are right where we are now. At this moment, it is very likely that we are not be able to say whatever we want to them, despite this, we are very happy with their own contributions to our project.<br />
<br />
:We have to recognize that, specially this people, brought us answers that we were not able to find by ourselves. For us, for our team, and for our proyect, the most important thing they did was their support that came together with very useful advices. <br />
<br />
:Our special thanks are principally to:<br />
<br />
'''-Dra. Sonia Vazquez Flores''' One of our principal advisors and encouragers to iniciate our team and project.<br />
<br />
'''-Ing. Rodrigo Machado''' Our lastest president and now recently graduated, and the most important thing, our friend who always has been there for supporting us. <br />
<br />
'''-Ing. David Maycotte''' Ex member of this team which we appreciate for his unconditional support.<br />
<br />
'''-Ing. Antonio Villarreal ''' Ex member of our team, actually now he is studying a mastery on Synthetic Biology in Paris, France. He helped a lot with the building of our construct.<br />
<br />
'''-Aldo Diaz''' Ex member of this team, and our friend. We appreciate that he always helped in every problem we told him.<br />
<br />
'''-Citlalli Rosas''' Director of "Grupos Estudiantiles" and principal advisor in our bureocratic procedures.<br />
<br />
'''-Dr. Gerardo Montejano''' Director of Food Industry, Biotechnology and Agronomist Engineering School. He always has been supporting us in whatever we need related with the Department.<br />
<br />
'''-Dr. Hector Morelos''' Director of Engineering and Architecture Division. He, just like Dr. Morelos did, supported and helped our project.<br />
<br />
<br />
'''-Dr. Alejandro Olmos''' He teaches Enzimology and Biocatalisis. He did very significant things for us, just like giving us very worth advises and also he could acquire L. Plantarum for our project.<br />
<br />
'''-Dr. Javier Rangel''' Molecular Biology and Biosensors teacher, that helped us at the begginig of our project solving some questions and bringing us ideas.<br />
<br />
'''-Ambey Shree Shukla''' Molecular Diagnostic and Functional Genomics teacher. She is a very polite person, always gentle with the team. She also always showed respect and interest for helping in the project.<br />
<br />
'''-Prashant Kumar Misrha''' Ex professor of Genetic Engineering. He was one of our first advisors, and no matter what, where, and when (even 2:00 a.m.) he was always for whatever we needed.</div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/HeaderTeam:BIOSINT Mexico/Header2013-09-28T02:41:57Z<p>Gabyqz: </p>
<hr />
<div><html><br />
<style><br />
<br />
body{<br />
background: #F2F0F0;<br />
<br />
<br />
}<br />
#p-logo{<br />
display:none;<br />
<br />
<br />
}<br />
#top-section {<br />
height:130px ! important;<br />
border: 0;<br />
width:100%;<br />
background: #bfd255; /* Old browsers */<br />
background: -moz-linear-gradient(top, #bfd255 0%, #8eb92a 50%, #72aa00 51%, #9ecb2d 100%); /* FF3.6+ */<br />
background: -webkit-gradient(linear, left top, left bottom, color-stop(0%,#bfd255), color-stop(50%,#8eb92a), color-stop(51%,#72aa00), color-stop(100%,#9ecb2d)); /* Chrome,Safari4+ */<br />
background: -webkit-linear-gradient(top, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* Chrome10+,Safari5.1+ */<br />
background: -o-linear-gradient(top, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* Opera 11.10+ */<br />
background: -ms-linear-gradient(top, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* IE10+ */<br />
background: linear-gradient(to bottom, #bfd255 0%,#8eb92a 50%,#72aa00 51%,#9ecb2d 100%); /* W3C */<br />
filter: progid:DXImageTransform.Microsoft.gradient( startColorstr='#bfd255', endColorstr='#9ecb2d',GradientType=0 ); /* IE6-9 */<br />
}<br />
<br />
#menubar{<br />
margin-left: 175px;<br />
}<br />
.right-menu li a {<br />
background-color: transparent;<br />
}<br />
#searchInput {<br />
float:right;<br />
height: 25px;<br />
width: 170px;<br />
font-size: 14px;<br />
border: 1px solid #000000;<br />
border-radius: 5px 5px 5px 5px;<br />
-webkit-border-radius: 5px 5px 5px 5px;<br />
-moz-border-radius: 5px 5px 5px 5px;<br />
box-shadow:inset 1px 1px 3px #000;<br />
-webkit-box-shadow:inset 1px 1px 3px #000;<br />
-moz-box-shadow:inset 1px 1px 3px #000;<br />
padding-left:5px;<br />
}<br />
input.searchButton{<br />
color: #ffffff;<br />
}<br />
input.searchButton:hover{<br />
color: #4682b4;<br />
}<br />
<br />
#footer-box a{<br />
color:#fff;<br />
}<br />
#content {<br />
background: #fff;<br />
border: 0;<br />
color: #333;<br />
margin: 50 auto;<br />
<br />
<br />
position: relative;<br />
width: 85%;<br />
z-index: 2;<br />
border-radius: 5px 5px;<br />
text-align: justify;<br />
<br />
box-shadow: 0px 0px 20px #333;<br />
-webkit-box-shadow: 0px 0px 20px #333;<br />
-moz-box-shadow: 0px 0px 20px #333;<br />
}<br />
#content img{<br />
margin: 10px;<br />
box-shadow: 0px 0px 0px #333;<br />
-webkit-box-shadow: 0px 0px 0px #333;<br />
-moz-box-shadow: 0px 0px 0px #333;<br />
}<br />
#bodyContent{<br />
width:95%;<br />
background:#fff;<br />
margin:0 auto;<br />
<br />
}<br />
<br />
.firstHeading {<br />
/*text-shadow: 1px 1px 2px #000;<br />
margin-left: 20px;<br />
color:#ff0000;<br />
font-size: 36px;<br />
font-weight: bold;<br />
font-family: Helvetica,Arial,sans-serif;*/<br />
display:none;<br />
}<br />
.myowntitle{<br />
text-shadow: 1px 1px 2px #000;<br />
margin-left: 30px;<br />
color:#008000;<br />
font-size: 36px;<br />
font-weight: bold;<br />
font-family: "Comic Sans MS", cursive, sans-serif;<br />
}<br />
h1{<br />
border-bottom:0;<br />
}<br />
<br />
#catlinks {<br />
background-color: transparent;<br />
border: 0;<br />
clear: both;<br />
margin:10px 0 ;<br />
padding: 10px;<br />
}<br />
#my_own_top{<br />
margin: 0 auto;<br />
position: absolute;<br />
top:-186px;<br />
width: 975px;<br />
height: 130px;<br />
overflow:hidden;<br />
}<br />
#my_own_top h1{<br />
<br />
color:#fff;<br />
font-size:50px;<br />
font-weight:bold;<br />
padding:0;<br />
height:50px;<br />
width:300px;<br />
margin:100px 30px 0 350px;<br />
padding-left:30px;<br />
position:relative;<br />
}<br />
#my_own_top img.logo{<br />
float:right;<br />
position:relative;<br />
}<br />
#my_own_top img{<br />
position:relative;<br />
}<br />
#navigation{<br />
float:left;<br />
width:100%;<br />
height: 40px;<br />
background:#000;<br />
margin-top:-20px;<br />
border-bottom-right-radius:8px;<br />
border-bottom-left-radius:8px;<br />
}<br />
#navigation ul{<br />
list-style-type: none !important;<br />
list-style-image: none;<br />
}<br />
#navigation ul li{<br />
width:130px;<br />
min-height: 35px;<br />
text-align:center;<br />
vertical-align: middle;<br />
position: relative;<br />
float: left;<br />
margin-right: 10px;<br />
background:#000;<br />
}<br />
#navigation a{<br />
text-decoration:none;<br />
font-size: 17px;<br />
color: #FFF;<br />
text-shadow: 1px 1px 5px #fff;<br />
}<br />
#navigation a:hover{<br />
color:#fff;<br />
background:#2E2E2E;<br />
font-size:1.4em;<br />
text-shadow:1px 1px 5px #81F7F3;<br />
}<br />
<br />
#navigation li:hover{<br />
background:#2E2E2E;<br />
<br />
}<br />
.menu_body {display:none; width:184px;margin-left: 0px; margin-top: 10px; z-index:999;}<br />
.menu_body li{background:#fffafa; background-image: none !important;z-index:999; margin:0; border-radius: 0px;}<br />
.menu_body li.alt{background:#fffafa;background-image: none; border-radius: 0px;}<br />
.menu_body li a{color:#fffafa; text-decoration:none; padding:10px; display:block;}<br />
.menu_body li a:hover{ font-weight:bold;}<br />
p{ font-style:italic; padding-left:20px;padding:right:20px; margin-right:20px;}<br />
<br />
<br />
<br />
</style><br />
<br />
<div id="my_own_top"><br />
<a href="https://2011.igem.org/Team:ITESM_Mexico" ><img src="http://s22.postimg.org/7n99nq2b5/A_lo_largo.png" alt="Biobrickoatl Home" class="logo" /></a><br />
<div style="float:center;position:absolute;" ><br />
<h1>SmartPro</h1><br />
<h1 style="font-size: 36px; color:#db0976; height:50px;">Smartpro</h1><br />
</div><br />
<a href="https://igem.org" ><img src="http://deviatan.com/sponsoring/igem_light.png" alt="Igem" style="margin:50px 100px 50px 0;"/></a><br />
<a href="http://postimg.org/image/uvvpixuvr/" ><img src="http://s23.postimg.org/rp15zbafv/Logotipo_Definitivo.png" alt="LogotipoDefinitivo" style="margin:30px 30px 30px 0;" /></a><br />
<br />
<br />
</div><br />
<br />
<div id="navigation"><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico">Home</a><br />
<br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Team">Team</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Who we are">Who we are</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Acknowledgment">Special Thanks</a></li><br />
<li><a href="https://igem.org/Team.cgi">Official team page</a></li><br />
</ul><br />
<br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Project">Project</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Abstract">Abstract</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Chassis">Chassis</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Sensor">Sensor</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Safety: Kill switch">Safety: Kill switch</a><br />
</li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Construction">Construction</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Results">Results</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Future research">Future research</a></li><br />
<br />
</ul><br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Parts">Parts</a><br />
<br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Modeling">Modeling</a><br />
</li><br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Human Practice">Human Practice</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Probiotics: iGEM contribution">Probiotics: iGEM contribution</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Workshop">Workshop</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Publicity">Publicity</a></li> <br />
<br />
</ul><br />
</li> <br />
<li ><br />
<a href="https://2013.igem.org/Team:BIOSINT_Mexico/Notebook">Notebook</a><br />
<ul class="menu_body"><br />
<li><a href="https://2013.igem.org/Team:BIOSINT_Mexico/Protocols">Protocols</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOINT_Mexico/Lab work">Lab Work</a></li><br />
<li><a href="https://2013.igem.org/Team:BIOINT_Mexico/Gallery">Gallery</a></li><br />
</ul><br />
</li><br />
<br />
</ul><br />
</div><br />
<script type="text/javascript" src="https://ajax.googleapis.com/ajax/libs/jquery/1.6.2/jquery.min.js"></script><br />
<br />
<script type="text/javascript"><br />
$(document).ready(function () {<br />
<br />
<br />
<br />
$("ul.menu_body li:even").addClass("alt");<br />
$('div#navigation ul li a').mouseover(function () {<br />
//alert(this);<br />
$(this).parent().find("ul.menu_body").slideDown('medium');<br />
$(this).parent().hover(function() {<br />
}, function(){<br />
$(this).parent().find("ul.menu_body").slideUp('medium');<br />
});<br />
});<br />
<br />
$('ul.menu_body li a').mouseover(function () {<br />
$(this).animate({ fontSize: "16px" }, 50 );<br />
});<br />
$('ul.menu_body li a').mouseout(function () {<br />
$(this).animate({ fontSize: "15px" }, 50 );<br />
});<br />
<br />
<br />
<br />
<br />
<br />
});<br />
<br />
</script><br />
<br />
</html></div>Gabyqzhttp://2013.igem.org/Team:BIOSINT_Mexico/ResultsTeam:BIOSINT Mexico/Results2013-09-28T02:39:53Z<p>Gabyqz: </p>
<hr />
<div>{{:Team:BIOSINT_Mexico/Header}}<br />
<br />
<br />
----<br />
<html><h1 class="myowntitle">Results</h1> </html><br />
----<br />
----<br />
<br />
[[File:Piezasgens.jpg|right|300px]]<br />
<br />
Plasmid extraction from synthetized parts <br />
<br />
pUC57: 2710 bp<br />
<br />
*In the lane 1 is the ladder of 5000 bp<br />
<br />
*The sample of the lane 3 is the promoter 1, inserted sequence: 103 bp<br />
<br />
*The sample of the lane 5 is the promoter 2, inserted sequence: 103 bp<br />
<br />
*The sample of the lane 7 is the RBS, inserted sequence: 76 bp<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
<br />
<br />
[[File:Piezas_PsB1C3.jpg|right|300px]]<br />
<br />
<br />
<br />
<br />
<br />
Parts clonning in pSB1C3: 2070 bp<br />
<br />
<br />
*In the lane 1 is the ladder of 3000 bp<br />
<br />
*The sample of the lane 3 is the promoter 2<br />
<br />
*The sample of the lane 4 is the promoter 1<br />
<br />
*The sample of the lane 7 is the RBS<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
<br />
[[File:Plasmid_GFP.jpg|right|300px]]<br />
<br />
<br />
<br />
<br />
<br />
Ligation Promoters with GFP<br />
<br />
<br />
*In the lane 1 is the ladder of 5000 bp<br />
<br />
*The sample of the lane 3 is the Plasmid GFP<br />
<br />
*The sample of the lane 5 is the Plasmid GFP</div>Gabyqzhttp://2013.igem.org/File:Bibriocoatl_black_background_by_humano_normal-d47e2ou.pngFile:Bibriocoatl black background by humano normal-d47e2ou.png2013-09-28T00:52:32Z<p>Gabyqz: uploaded a new version of &quot;File:Bibriocoatl black background by humano normal-d47e2ou.png&quot;</p>
<hr />
<div>hola</div>Gabyqzhttp://2013.igem.org/File:Bibriocoatl_black_background_by_humano_normal-d47e2ou.pngFile:Bibriocoatl black background by humano normal-d47e2ou.png2013-09-28T00:48:59Z<p>Gabyqz: hola</p>
<hr />
<div>hola</div>Gabyqzhttp://2013.igem.org/File:Leaf-cells-wallpapers_10681_1680x1050.jpgFile:Leaf-cells-wallpapers 10681 1680x1050.jpg2013-09-28T00:46:41Z<p>Gabyqz: uploaded a new version of &quot;File:Leaf-cells-wallpapers 10681 1680x1050.jpg&quot;: Reverted to version as of 00:45, 28 September 2013</p>
<hr />
<div></div>Gabyqz