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CAROL
Gel Extraction of luxCDABE
- The main purpose of gel extraction is to ensure that only luxCDABE is present for cloning. The problem with cloning luxCDABE is that we are cloning in smaller pieces of DNA, rather than the gene of interest.
- Prepared 0.7% gel and ran the gel for 90 minutes at 90V.
- Followed QIAquick Gel Extraction Kit Protocol (please see protocol page for details)
- The concentrations that we ended up getting from the gel extraction is low. We re-ran the 0.7% gel with a larger amount of product, however, the kit is only optimal for 3-4 kbs.
- Gradient PCR of luxCDABE with the biobrick sites at the end. (We ran out of PCR products to work with)
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CHINMOYEE
Biosafty 1 Course
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EMILY
Visualization of Colony PCR and Retreaks
- Today I started with re-running my gel from Friday from the PCR products in the freezer. See gel photo below.
- Analysis: Lanes 1-4 are J13002-LuxOD47E-B0015 trial 1 (with the J13002-LuxOD47E construct as the insert) and Lanes 5-8 are J13002-LuxOD47E-B0015 trial 2 (with B0015 as the insert). Lane 9 is a size control with J13002-LuxOD47E and Lane 10 is a size control with only J13002. If our transformation was successful, we expect to see lanes 1-8 slightly higher then lane 9, which should be slightly higher than lane 10.
- Looking at this gel, we do not see a size difference at all between lanes 1-8 and lane 9. This indicates that either our transformation was not successful or our initial contruction digest was not successful. Because we have all ready gone back to the unligated product once with no results, we will try this again going back to contruction.
- Today I also made restreaks of the J13002-Lux0D47E constructs on KT1144 plates as well as overnight cultures of J13002-LuxOD47E and LuxOD47E witht he purpose of making glycerol stocks tomorrow.
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FAHD
Descriptive Title of What You're Doing
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IMAN
Descriptive Title of What You're Doing
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JAMIE
Descriptive Title of What You're Doing
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JEREMY
Descriptive Title of What You're Doing
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KATIE
Descriptive Title of What You're Doing
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KEVIN
Construction of Pqrr4+I13500 (RBS and GFP)
Now that I13500 part has been confirmed working by putting R0040 infront, we can move on to the construction of our reporter circuit, which has Pqrr4 promoter instead of R0040. Because the Pqrr4 promoter is in AC plasmid and I13500 is in A, Pqrr4 was cut with SpeI+PstI, and I13500 was cut with XbaI+PstI. The vector, Pqrr4, was phosphotase treated, and then the two parts were ligated together. Next, the product was transformed into TOP10 cells and were plated on AC plates. Overnight growth is needed.
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MANDY
Descriptive Title of What You're Doing
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PATRICK
Descriptive Title of What You're Doing
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PRIMA
Mini-prep -Lux PQ in Ak
Purpose: Isolate plasmid from Lux PQ in Ak. PQ-B-R-OU-B, aiiA, C1 lambda
Procedure: used Qiagen mini-prep directions. The following table shows the labeling scheme.
| Plasmid | 260/280
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| Tube numbers | Contents
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| 1 | Lux PQ in AK colony 2
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| 2 | Lux PQ in AK colony 5
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| 3 | Lux PQ in AK colony 6
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| 4 | Lux PQ in AK colony 8
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| 5 | PQ-B-R-OU-B Colony 11
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| 6 | PQ-B-R-OU-B Colony 12
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| 7 | PQ-B-R-OU-B Colony 13
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| 8 | C1 lambda Colony 1
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| 9 | aiiA colony 1
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| 10 | aiiA colony 2
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| 11 | aiiA colony 3
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| 12 | aiiA colony 4
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We used buffer EB (heated at 70 degrees) because it's easy to dilute plasmid for larger pieces. Note: when nano-dropping, Blank with EB b/c i eluted with EB buffer.
Today, I also learned how to look up which enzymes to use in restriction digest using the buffer-enzyme sheet.
Following table shows nano dropping results:
- Results: Used Nanodrop 1000 Spectrophotometer read at 260 wavelength to determine DNA concentrations
| Plasmid | 260/280 | 260/230 | Concentration [ng/μL]
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| Lux PQ psB1AK3 C2 | 1.83 | 2.20 | 102.7
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| C5 | 1.82 | 2.33 | 92.7
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| C6 | 1.84 | 2.07 | 101.7
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| C8 | 1.85 | 2.15 | 113.8
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| PQ-B-R-OU-B C11 | 1.83 | 2.09 | 145.2
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| C12 | 1.85 | 2.15 | 125.7
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| C13 | 1.87 | 2.01 | 157.4
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| C1 lambda psBAK3 C1 | 1.75 | 1.54 | 63.3
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| aiiA psB1A2 C1 | 1.68 | 0.77 | 35.9
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| aiiA C2 | 1.71 | 1.73 | 30.7
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| C3 | 1.70 | 31.6
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| C4 | 1.84 | 2.39 | 28.6
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STEFAN
Reading ethics papers
Today I read 2 ethics papers:
Erik Parens, Josephine Johnston, and Jacob Moses, “Ethical Issues in
Synthetic Biology: An Overview of the Debates,” Woodrow Wilson
International Center for Scholars, June 24, 2009.
Selgelid M., 2007, The tale of two studies: Ethics, Bioterrorism, and
the Censorship of Science, Hastings Center Report 37, no. 3:35-43.
and took some notes!
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VICKI
Systems biology textbook
This is a slight diversion from the other work, but it does provide an intersting spin on what we're doing. This is really interesting as I’ve seen most of what Alon discusses but always in different contexts. I was distracted for a while on feedforward control (which is fascinating in its applications, by the way). I’m working through it right now.
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