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Team:XMU Software/Notebook
From 2013.igem.org
3.30.2013
Chen: Experimental workspace can automatically decide whether it is a personal account or a business account of networking and the workspace affords the download link of those relevant software. Can we get those software embedded? How to help our customers find out the software they want quickly? (By searching them?) It is also important to process data and generate lab reports and logs in the workspace.
Future plan: We plan to add software in the software library, optimize the software's functions and landscaping interface. Besides, we want to afford real time data while monitoring on different platforms. And how about syncing to the cloud?
Qiu: Easy Express. The software is designed to save time for users. Then they just need to stay on creating new ideas and putting a possible idea into practice. In addition, unified expressions get researchers to understand other's work easily. Its system's function: drawing, display sequences, dynamic display structure of the plasmid and parts of the experiments, shows the meaning of each graph and table, guiding and help. The challenge for us is that whether we can dynamic display the data? If we standardization of the presentation, the software may be rejected by other teams for its boredom standardization separate kinds of efforts may be impossible. This software told you that you don't need to learn MAYA and other magic software, it can include their functions. Will it be possible? (Perhaps unattainable for now) The idea may be realized by attaching the database to the software and affording individuation information search for our users.
Wang: Document management and real time monitoring can help you on scientific literature management, take notes and share your ideas. It can also automatic detection and data recording of streamlined repetition, design shortcut keys to improved operational efficiency. Thus, users can use graph visualization to understand the data easily.
Zhang: She had done a lot of work on directional optimization of BioBricks.
Purpose: Inputing environmental parameters include special loci genes, the expected expression level and chooses candidate BioBricks. Optimize and find out the best sequences under given conditions and setting number of evolutionary (n) chooses the BioBricks, and the system will guide users until the given conditions are met, variation to generate different sequencess. Copy, retain or reject BioBricks according to the matched-degree, this step is parallel to natural selection. Circulation stops after n times circulations, output the best sequences. The problems are how to make sure that the genotype equals its phenotype? How to define the f(x) that we want? And now we need to read some books about the genetic algorithm to get the way.
Summary: Now we have two ideas. One is to build a platform for researchers that focusing on synthetic biology (aim to increase their efficiency). Former researchers have already done some researches about it, so it might be a rat race.
Solve synthetic biology problems with GA. We need to supplement relevant basic knowledge of GA. So, studying CLOTHO developed by Berkeley and other universities and try to find out some flesh ideas and make a research to find out what front-line researchers need. We also need to study pertinent software and GA, protein quaternary structure and knowledge about directional optimization, read more books and literature to make sure the possibility of the idea. GA is too difficult for some of us. We still need to get come up with some novel ideas.
OK, for so many tasks, work in groups and share additional ideas~!
2013.4.7
Zhang had learnt GA. Also, we read the news about Calgary and Newcastle, SUSTC（calculate terminator efficiency by a score system）. Code is linked up with fitness, which influences the secondary structure of protein. For example, the changing of a-tail, stem loop and T-tail, we can also find it in the project of SUSTC. Additionally, she also read papers on how to calculate f(x) & take notes when learn new things. SUSTC' fault: Just one function, it may not catch everybody's eyes. She plans to change promoter so as to confirm the system efficiency. Jianxing schedule to learn neural network, after that, he will change the source code to be used on today's system. Huang Xin and Huang Jian now know just a little about Matlab, but they think the code can be changed easily. Wang learned Matlab as well. Tang Chun read some books about system efficiency and downloaded papers to share with others. From N-terminal domain rule, he noticed that choose the parameters is a key point for our project.
Next week's task had been written on the wall. And we suggest everybody to use PowerPoint to explain their ideas.
2013.4.14
Specific our goals: For most of us aren't familiar with GA. We misunderstand the idea at the beginning. We determined to find the best fit BioBricks through genetic recombination and variation inside the BioBricks, and we evaluating them by our algorithm. Compare with other team' work: EbolGENTS (2008, Calgary) and Evolutionary Algorithm (2008, Newcastle University). Both of them had used GA, however, they construct a system with given BioBricks just like build blocks, after which they find the best system in those "structures". As for our group, we focus on genetic recombination and variation inside the BioBricks to make further ones, and search for the best with given conditions.
Reduce the degree of difficulty: it may be a huge project to ensure phenotype and fitness result from mutations of the base sequences in the whole BioBricks. So we decided to narrow down and aim at optimizing the decision of particular bases, promoter, terminator and RBS locus. Yijuan had mentioned that SUSTC had done research on computational efficiency of the Terminator's gene sequences.
Core problem: How to ensure phenotype from genotype? How to find the f(x) meet our goal? Fitness function: three ways had been chosen to solve it: first, search for relevant literatures; read the project offered by Professor Fang named “microorganism’s choice of the optimal conditions"; if possible, our wet lab should give us a hand on affording real time data and experimental simulation to ensure f(x).
Current task: Check the project of iGEM "calculate terminators efficiency by the sequences of the terminators”; all of us need to take a look at SUSTC' work and discuss the next time. Learning Matlab and GA!
Annotations: We had set sail now! All of us need to learn SUSTC's work on ensuring genotype by phenotype and discuss the next time. We are badly in need of professor's help!
Try to find more idea! Stay hungry!
2013.4.21
This week we had a summary of the work we had done before and nowadays we need to fix it. GA as an algorithm can work on several biological questions. And several new ways and improvement had been found out to solve the problem. Take an example, GASCO is a new algorithm that improves the answer of GA.
We start the preparation for the human practice. That may be of great importance. We had to distribute the job and explicit the goal so as to lift efficiency.
Neural network can be used in it.
2013.4.28
Zhang: After reading several papers, she got down to SBOL. She got some new ideas on our program.
Han Tao & Huang Jiang & Huang Xin: They had successly got a software that can simulate the situation. However, software remains some problem to be solved. We had better make a new one, do not use this one, for we can use Matlab. That will be a good idea.
Platform and relevant work went well.
General Protocols
1 Stock solution
50 mg/mL Ampicillin
-0.5 g Amp, 10 mL water, filter sterilize with millipore express membrane, freeze in aliquots
50 mmol/L Arabinose
- 0.1876 g Arabinose, 25 mL water, filter sterilize with millipore express membrane.
2 Preparation of Competent Cell
Thaw an aliquot of cells (without any plasmid in them) on ice
- To 20 mL of sterile LB, add 100 μL aliquot of the thawed cells: remember, this LB does not have any antibiotic in it, so work as aseptically as possible (i.e. autoclave all solutions and use sterile pipettes).
- Grow cells in the shaker at 37 ℃ and 200 rpm, until they reach an OD600= 0.3 - 0.4. This usually takes 1.5 - 2 hours.
- Ice down the LB with growing cells for 30 min.
- Aliquot into sterile 1.5 mL tubes and spin down at 1500 xg for 5 min at 4 ℃; discard supernatant.
- Ice down Solution A and Solution B of TaKaRa Competent Cell Preparation Kit during centrifugation.
- Gently resuspend each pellet with 100 μL Solution A.
- Centrifuge 1500 xg for 5 min and discard supernatant.
- Resuspend each pellet on ice in 100 μL Solution B.
3 Transformation
- Add 10 μL of DNA. Swirl gently with pipette.
- Incubate tubes on ice for 30 min
- Heat pulse tubes in 42 ℃ water bath for 90 s.
- Incubate on ice for 5 - 10 min
- Add 400 μL of LB broth to each tube and incubate for an hour at 37 ℃ with shaking.
- Spread 100 μL of each culture on an LB agar plate containing the appropriate antibiotics and incubate overnight at 37 ℃ (spread using beads).
4 Plasmid Purification
- Centrifuge sample in eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution.
- Resuspend the pelleted cells in 250 μL of the resuspension Solution (mixture with Solution I and RNasa A). The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
- Add 250 μL of the Lysis Solution (Solution II) and mix thoroughly and gently by inverting the tube 5-6 times, letting it stand for 1-2 min at room temperature until the solution becomes viscous and slightly clear.
- Add 350 μL of the Neutralization Solution (Solution III) and mix immediately and thoroughly by inverting the tube 5 - 6 times.
- Centrifuge for 10 min at 12,000 rpm to pellet cell debris.
- Apply the supernatant to the supplied spin column by decanting. Avoid disturbing or applying the white precipitate.
- Centrifuge for 1 min at 12,000 rpm. Discard flow-through and place the column back into the same collection tube.
- Add 500 μL of the Wash Buffer PB to the spin column. Centrifuge for 1min at 12,000 rpm and discard flow-through. Place the column back into the same collection tube.
- Add 500 μL of the Wash Buffer W to the spin column. Centrifuge for 1min at 12,000 rpm and discard the flow-through. Place the column back into the same collection tube.
- Repeat the step 9 again.
- Discard flow-through and centrifuge for an additional 3 min to remove residual Wash Solution.
- Place the spin column in a clean 1.5 mL centrifuge tube, and pipet 20 μL Elution Buffer TE (prewarm to 60 ℃) directly to the center of the column without touching the membrane. Let it stand for 2 min at room temperature and centrifuge for 1 min at 12,000 rpm.
- Discard the column and store the purified plasmid DNA at -20 °C.
5 Standard BioBrick Assembly
- Digestion of insert: 2 - 5 μg DNA / 100 μL volume, 10× H buffer, EcoR I, Spe I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try avoiding staining or exposure to ultraviolet light of the insert.
- Digestion of vector: 2 - 5 μg DNA / 100 μL volume, 10× M buffer, EcoR I, Xba I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.
6 Suffix Insertion
- Digestion of insert: 2 μg~5 μg DNA / 100 μL volume, 10× M buffer, Xba I, Pst I. Digestion and inactivation. Clean up the insert.
- Digestion of vector : 2 μg~5 μg DNA / 100 μL volume, 10× H buffer, Spe I, Pst I. Digestion and inactivation. Clean up the vector.
7 Ligation
- After digestion and clean-up, the next step is ligation.
According to the ligation equation of Insert/Vector=3 mol/mol, combine the vector, insert, 10× T4 ligation buffer and 1 μL of T4 ligase in a 10 μL reaction overnight at 4 ℃.
8 Gel Extraction
- Weigh a 1.5 mL centrifuge tube for each DNA fragment to be isolated and record the weight.
- Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close as possible to the DNA to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 mL tube and weigh. Record the weight of the gel slice.
- Add Bing Buffer BD at a ratio of 100 μL of solution per 100 mg of agarose gel slices.
- Incubate the gel mixture at 55-65 ℃ for 7-10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved.
- After the dissolved gel mixture cool down, transfer it to the Spin Columns assembly and incubate for 2 min at room temperature.
- Centrifuge the Spin Columns assembly in a microcentrifuge at 12,000 rpm for 1 min, and discard the flow-through.
- Wash the columns by adding 500 μL of Wash Buffer PE to the Columns. Centrifuge the columns assembly for 1 min at 12,000 rpm, and discard the flow-through.
- Repeat step 7 again.
- Centrifuge the Columns for an additional 3 min to completely remove residual wash buffer.
- Empty the Collection Tube and recentrifuge the column assembly for 1 min with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.
- Place the spin column in a clean 1.5 mL microcentrifuge tube, and pipet 20 μL deionized water (pH is 8.0 - 8.5 and prewarm to 60 ℃)directly to the center of the column without touching the membrane. Incubate at room temperature for 2 min.
- Centrifuge for 1 min at 12,000 rpm. Discard the columns and store the microcentrifuge tube containing the eluted DNA at–20 ℃ .
9 Restriction analysis
- Combine H20, 4μL of dNTP Mixture, 10×Ex Taq Buffer, 0.25μL of Ex Taq, 1 μL of VR primer, 1 μL
of VF2 primer and pick the colony in a 25 μL reaction for PCR.
- Gel electrophoresis.
10 Genetic mutations
1. Design the primers with mutation.
2. PCR.
- 0.25 μL of Pyrobest DNA Polymerase (5U/μL).
- 5 μL of 10 x Pyrobest buffer II.
- 4 μL of dNTP Mixture (2.5 mM each).
- 1 μL of Primer 1 (20 uM).
- 1 μL of Primer 2 (20 uM).
- 0.01 – 1 ng Template.
- Add ddH2O up to 50 μL.
3. PCR condition.
- 94 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 5 min . circulate for 24 times.
4. Run the gel and do the gel extraction.
5. Blunting Kination.
- 1 pmol of DNA Fragment.
- 2 μL of 10 x Blunting Kination Buffer.
- 1 μL of Blunting Kination Enzyme Mix.
- Add ddH2O up to 20 μL.
- Reaction under 37 ℃ for 10 min.
- Reaction under 70 ℃ for 10 min.
6. Ligation.
- Take 5 μL reacting solution into a new PCR tube.
- Add 5 μL Ligation Solution I.
- Reaction under 16 ℃ for 1 h.
- Transfor the reacting solution into 100 μL competent cell.
Characterization
Fluorescence Measurements
- The samples to be tested are cultured from plates in 20 mL of the Basal Minimal Medium with appropriate antibiotics and incubated overnight at 37 ℃ at 200 rpm.
- The culture is checked for OD600 next day and then subculture by the same medium with antibiotics at 37 ℃ shaking for 2 h.
- Add corresponding inducer at concentration gradients into the above-mentioned culture and keep on incubating. During the time incubating, every 15 min, take 1 mL bacteria liquid, then centrifuge the cells( 6000 rpm, 10 min ) and resuspend them in 1 mL PBS. At last, pipette to a 96 well plate.
- The plate reader made by Molecular Device then read.
- The program does the following:
- In endpoint reads, following measurements are taken in a time interval of 15 min: absorbance (600 nm filter) and fluorescence (485 nm and 520 nm for GFP).
- The results then transfer to excel sheet and interpret.
