May Crash Course in Synthetic Biology
In order to get some serious training uner our belt, do something that helps iGEM, and start work that can be credited towards our project we will be testing Biobrick promoter strength on May 6-10th.
- Request Biobrick promoters that previous teams have made but that have not yet received adequate characterization. (To be done NTL April 25)
- Perform assembly of promoters into plasmid vectors.
- Transform E.coli with the constructed plasmids and grow up colonies.
- Measure GFP expression of test promoter against reference standard.
To do this training, make sure that you are familiar with "Measuring the activity of Biobrick Promoters Using an In vivo Reference Standard" by Jason Kelly et al.. The supplementary materials are also important and can be safely downloaded by clicking onthis link here. For a graphical summary of the protocol, travel over to the Parts Registry Measurement Section. (The images are also in the supplementary materials.)
By measuring these parts against a standard, we'll get a tremendous amount of experience in many of the protocols we'll need to have mastered for summer. Plus, we could submit our results to the Registry and have already contributed before the summer even starts.
- Please note that we WILL NOT be doing Step #1 (synthesis and annealing of oligos to make a test promoter). Instead, we will be testing promoters that have already been made by previous iGEM teams. This will save us money and it helps iGEM out to have this QA performed.
TK's Restriction Digest
Materials
- Restriction enzymes (EcoR I, Spe I, Xba I or Pst I) from NEB
- NEB2 buffer
- BSA
- Deionized, sterile H2O
Digest Mix
Example - 50 μL reaction. 100 μL reactions are also common especially if your DNA to be cut is dilute.
- 5 μL NEB2 buffer (for all digests with BioBricks enzymes, we use NEB2 buffer. It keeps things simple and seems to work).
- X μL DNA (usually ~500 ng depending on downstream uses).
- 0.5 μL 100X BSA (added to all digests because BSA never hurts a restriction digest)
- 1 μL BioBricks enzyme 1 (regardless of the volume of the reaction, 1 μL enzyme is used because generally this represents a 10-25 fold excess of enzyme and is therefore sufficient for most digests. Also, it can be difficult to accurately pipet less than 1 μL of enzyme since it is sticky due to the glycerol content.)
- 1 μL BioBricks enzyme 2
- (42.5 - X) μL deionized, sterile H2O
Procedure
1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
2. Add restriction enzyme buffer to the tube.
- Vortex buffer before pipetting to ensure that it is well-mixed.
3. Add BSA to the tube.
- Vortex BSA before pipetting to ensure that it is well-mixed.
4. Add appropriate amount of DNA to be cut to the tube.
- Vortex DNA before pipetting to ensure that it is well-mixed.
5. Add 1 μL of each enzyme.
- Vortex enzyme before pipetting to ensure that it is well-mixed.
- Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting.
6. Place in thermal cycler (MJ Research, PT-200) and run digest protocol.
- 4-6 hour incubation at 37°C
- Use a longer incubation time if you have time or are worried about the efficiency of cutting. I think this time can be shortened to 2 hrs while still cutting to completion.
- 20 mins at 80°C to heat inactivate enzyme.
- This step is sufficient to inactivate even Pst I.
- 4°C forever (or until you pull the reaction out of the thermal cycler).
7. Generally, use some method of DNA purification to eliminate enzymes and salt from the reaction.
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