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Team:Baltimore US/Notebook
From 2010.igem.org
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Group NotebookNotes for October 2010
June 29th, 2010In attendance: Day - Duke, Tom, Patrick, Ryan, Robert Convert to Bb format - Add Bb Prefix and Suffix 2. Characterize Part Error Rate? 3. Educational Supplementation Techniques - How to do a single point mutation
Identifying parts available. What is a Neural Network? The XOR switching system. - Addressable Conjugation.
June 28th, 2010In attendance: Day - Duke, Robert, Patrick, Robert, Ryan, Tom June 24th, 2010In Attendance: Patrick, Robert, Tom, Duke, Ryan June 16th, 2010In attendance: Lisa, Ryan, Tom, Colin (for a short time), Duke (for a moment) Earlier in the day, Robert and Ryan performed a mini-prep of the pcr'd plasmid backbones 3A1(2) and 5A1(2).
Robert also began working on finding optimized conditions for pcr. With the small turnout, Ryan began the digestion restriction with Colin's assistance. We combined the 2 strains of 3A1 and 5A1 together, then put together 3 - .8 eppendorf's with 50 ul of rxn ingrediates. After preparation, the cells were put into the pcr block for a 30 minute heat cycle. June 15th, 2010In attendance: Patrick, Ryan, Duke, Tom, Steve Transformation reaction - (Except we ran positive controls successfully)
Previously in Genetic Engineering all parts and pieces had variorus resistances where as bb tries to create a standard. Cells can be lost in replication due to metabolic costs, and new generations lacking the Costly programming tend to outgrow the special cells. When designing the assembly strategy try to use a plasmid backbone with a different resistance to allow the filtration/screening of parts that didn't transform correctly. Gel-Electrophoresis Prep Review for tonights run. Agarose 2 % for smaller parts will create a more viscous environment. 50ml final volume Weigh out the agarose (.4 gms for the 0.8%) (1 gm for the 2%) Emphasize again the importance of NCBI databases... http://www.ncbi.nlm.nih.gov/ June 14, 2010In Attendance: Tom, Duke, Patrick, Robert, Steve, Gary, David, Ryan, Miles 3 Separate transformations attempted: <partinfo>pSB1C3</partinfo> [1-3A] (ChlorAmphenacol resistant), <partinfo>pSB1K3</partinfo> [1-5A](Kanamicin resistant), <partinfo>pSB1T3</partinfo> [1-7A] (Tetracycline resistant) Control Group: PET-17B (still had separate resistance for Ampicillan) Tom will be culturing some adjacently resistant control groups for our next trial, thereby eliminating the extra variable in our testing. Procedural experience: 9 Parts for PCR - 7 Distributed amongst us. Various participants were asked to look up their function and various bp lengths. Patrick - <partinfo>Bba_R0010</partinfo> - 200 bp PCR for part [http://partsregistry.org/wiki/index.php/Part:BBa_R0063 R0063]: PCR Polymerase Chain Reactions Requirements Primers are 18-30 bp long. Have to be at least 15 bp long. Primers bind to template DNA, one primer for each (5'/3' strands). Review: What is a base..? Sugar with A, C, T or G is a nucleotide. BP are the matching set of 5' and 3' hydrogen bonded nucleotides. Polymerase links free dNTP's to the opened strand edges of basepairs. As the DNA heats it denatures then as it cools the primers anneal and polymerization takes place creating twice as many strands. The strands are held together only by hydrogen bonds, making it very easy to melt and reform. The polymerase acts like a little machine stitching the free dNTP's into the template. BioBrick plasmids have bb prefix and suffix. Primer binding sites vf2, vr. Theoretically any area can become a primer site. With PCR we can identify and amplify particular strands/compositions. You have to know how much of the requirements to use. Gary asked if Tom could show us where to find the optimized volume information at, and he agreed to pull out the research to show how to optimize. With PCR you can also design primers and introduce mutations. If primers are too short they can bind to the wrong spot. Actual reactions may vary based on bp length and combination. Usually they are tweaked for the optimal over a series of trials. PCR Reactions (Specific to this evening's trial)
34mM of dried DNA in primers ($12.50) Several companies provide these primers: invitrogen is an example. 34 x 10-9 moles / 100 x 10-6 liters = 3.4 x 10 -7 moles/liter Tomorrow we will look at the parts we pcr'd and then see how the bp length matches. If nothing happens its probably a pipetting error. If too many bands it may be an annealing issue. We will also look at the cells we plated of the various transformed parts, 3A, 5A, 7A, and see again if any of the colonies grew. June 10, 2010In attendance: Duke, Gary, Robert, Patrick, Ryan, Miles, Steven, Liz, and Colin. Tonight we tested the hypothesis that the plasmid did not code for the antibiotic resistance/that we did not use enough DNA. A Restriction Digest reaction was set up on the ligation reactions completed on June 7. The reactions were cut with EcoR1 and Pst1. The reactions were allowed to incubate at 37 degrees C for one and a half hours. The digests were then ran on a electrophoresis gel. No DNA was present as there was not enough DNA, thus proving Duke's hypothesis.
June 9, 2010In attendance: Duke, Tom, Ryan, Patrick, Lisa, Steven. Missing: Kyle and Friend(Forgot his name) 1) Registration for iGEM site & Team affiliation, required to update team wiki. 2) Access and update wiki.. Consider stylization, as well as information content. Who is our audience? Team info - blurb and caption of team members. Couple of sentences about you and your interests. Lab Notebook? - Openwetware connection. 3) Research biobrick parts by accessing in Registry of Standard Parts. XF to see if new combination are already on file? If not begin documentation of the 3 new assemblies, with image using standard BioBrick icons. Document bp length of new parts to compare with Gel-Electrophoresis. How do we test efficacy? (Part for PPM still in brick.) Is the media okay, is the antibiotic right. Positive tells you whether the cells themselves were capable of taking up our DNA. (Ideally, they should use the same antibiotic resistance, as our parts. Negative groups tells us whether the antibiotic and media was effective and that the cells were not resistant to the media. We know that cells were competent they were able to be transformed, and the antibiotic was effective. So the remaining question is whether they were able to be transformed. The ligation's are in question. Generally the restrictions and ligation's go smoothly. Tom's Hypothesis is questioning whether the media plates may have been mis-labeled with the wrong antibiotic resistance. Since we used a different resistance for controls, there was no telling. Possible denaturing of enzymatic proteins from the heat shock on the initial restriction? 80c kill cycle. Vector ? - Linear plasmids - was the amount to small...? Parts - size already was confirmed, according to Tom No Transformations.... so now what? Primers still on the way. Don't need to go to scratch.
Project idea: Basic tools/measurements
Lac polymerase - DIY-GEM How do you purify? What do you do with it... what is the process to create a project? Hardware only takes you so far, these 5 basic tools. Restrictions Ligations Transformations/Plating PCR Gel Electrophoresis The core will be good technique with these processes and then the ability to understand the existing database navigation with the proper questions to yield an experiment of interest. Research educational tools from the MIT educator on IGEM site. Should we have an e-mail/Comments section added to wiki - openwetware, so observers can ask us questions?
June 8, 2010In attendence: Colin, Patrick, Liz, Ryan, Duke and Tom. Missing: Andy, Scott. So tonight we came in and took the 4 ligation's/new part combinations from yesterday and transformed them into the competent cells, using a heat-shock transformation, while also preparing the Control groups (one with nothing/one with the ampicillin resistance). 3 of the parts were Tetracycline resistant, and 1 was Chlorephenecol. After the 90 minute transformation cycle we plated 6 versions of each of the 3 parts along with 1 plate of control and one negative group. The 6 plates were done in a 0, -1, -2 dilutions in 2 concentrations one of 50 ul, and one of 200 ul. They will be left for tomorrows team to run gel electrophoresis to determine whether they have the appropriate combination links.
June 7, 2010In attendance: Colin, Patrick, Robert, Ryan, David, Gary, Miles, Duke and Tom. Missing: Melissa. Tom greeted us with 4 separate sheets that contained 3 reactions each for us to begin restrictions and began heating to let the enzymes cut. A lesson in what not to do, was offered as we began the heating cycle of restriction in the PCR blocks and he timing had been set to 35 seconds instead of 35 minutes, after which it heated to a kill cycle of 80 degrees and we had to reapply the enzymes, in case the enzymes had been denatured. We had 6 individuals building the 4 sheets, 2 in redundancy. The legend for the various parts is as follows...
Patrick cut parts <bbpart>BBa_r0063</bbpart> and <bbpart>BBa_p0412</bbpart>, and the plasmid backbone <bbpart>pSBIT3</bbpart>. After which we had a round table discussion about what kind of projects we may follow up with and the process of using the NCBI databases to discover pre-existing sequence information related to our various ideas. One idea we have discussed was the option of creating a smoother introductory curve for fellow DIY-Bio commmunity members and the creation of home-brewed enzymes that might be to pricey for the amateur scientists. We ended the night with the beginning of the various ligation reactions, as seen above.
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