The overall goal of the synthetic biology protocol is to rapidly determine terminator functionality and quantify terminator strength in an in-vitro system that utilizes golden gate assembly. This protocol is used to test terminators that we found in silico using bio-informatics and test them in a biological setting. This allows for rapid and simple confirmation of bio-informatically identified terminators in a lab bench setting.
The main advantage of this protocol is its speed. We can go from bio-informatic identification to biological confirmation in less than one week. And in doing so, we can also verify that a terminator is in fact a terminator and we can quantify its terminating strength.
Generally, individuals will initially struggle with this technique, because it sounds counterintuitive to add the restriction enzyme with a ligase in the same tube. However we use type 2S restriction endonucleases and the recognition site for the enzyme is separate from the actual cut site. Once the terminator is ligated into the pGR blue plasmid, this is a permanent ligation.
However, by having the restricton enzyme and the ligase in the same tube, going through multiple cycles, we can achieve a high cloing efficiency. Begin this procedure by re-suspending the individual oligonucleotides in nuclease-free water at a concentration of 100 micromolar, then prepare 10x annealing buffer. Next, label a 1.5 millilter micro-centrifuge tube for each terminator to be tested.
To each tube, add 16 microliters of ultra-pure water, 2 microliters of 10x annealing buffer, and 1 micro liter each of the top and bottom strand oligonuclotides from the desired terminators. It's very important to make sure that the oligonuclotides you ordered have the correct sticky ends. Because once they're annealed together, it is important to ensure that the terminator is in the correct orientation.
If the terminator's in the wrong orientation, pGR blue will not be able to verify if it's a functioning terminator. Next place the micro-centrifuge tubes in a float and boil them for four minutes in a water bath. After four minutes, turn off the heat plate but leave the tube in the water overnight to slowly cool.
Store the annealed terminators at 20 degrees Celsius. To make a 40 nanomolar dilution of the annealed terminators, first thaw them on ice, then in a new 1.5-milliliter micro-centrifuge tube, add 124 microliters of nuclease-free water, to 1 microliter of the annealed terminators. Centrifuge the samples for 30 seconds at 10, 000x g.
After the spin, store the samples on ice. In a new thermocycler appropriate tube, add 6 microliters of nuclease-free water. Then add one microliter of commercial DNA ligase buffer.
Perform all of the subsequent steps on ice. Next, add 1 microliter containing 35 to 50 nanograms per microliter of the pGR blue destination plasmid, 1 microliter of the 40 nanomolar annealed terminators, 0.5 microliters of commercial high-fidelity BSA1 restriction endonuclease, and 0.5 microliters of commercial DNA ligase. Centrifuge the reaction mixture at 10, 000x g for 120 seconds.
Then place the tube directly in the thermocycler and run the following program. 20 cycles of 1 minute at 37 degrees Celsius, followed by 1 minute at 16 degrees Celsius and then one cycle of 15 minutes at 37 degrees Celsius. Following the run, either freeze and store the samples at 20 degrees Celsius, or proceed immediately to transformation.
Transform the ligation reactions into chemically competent e. coli cells using any appropriate transformation protocol. Here, JM109 cells are transformed using zippy transformation.
Using an L-shaped glass hockey stick, spread 25 microliters of the transformed cells onto pre-warmed LB agar plates containing 1 millimolar ampicillin, and 10 millimolar arabinose. Incubate the plates overnight at 37 degrees Celcius. Do not incubate them for more than 24 hours.
Ampicillin degrades rapidly. The next day, inspect the color of the colonies. A successful ligation should be white or yellow in visible light, and will fluoresce green under blue or UV light.
An unsuccessful ligation will produce a colony that is blue in color under visible light. For each of the terminators and controls, prepare a test tube containing 5 milliliters of sterile LB broth with 1 millimolar ampicillin and 10 millimolar arabinose. When performing this protocol, with pGR it's really important to ensure that the proper controls are in place.
The first control should just be LB broth. The next control should be cells that have resistance to ampicillin, but do not fluoresce under blue light. These will serve as a blank.
The next control is a pGR plasmid that is not cut so it has no terminator in place and this will serve as the baseline for terminator strength and the last one of your tubes will be for your sample. Under visible light, use a sterile loop to pick 2 to 3 white or yellow colonies, and transfer them to the LB broth. Allow the samples to incubate on a shaker at 37 degrees Celsius at 160 rpm for 16 to 18 hours.
The next day, use a spectrophotometer to determine the optical cell density of each sample at 600 nanometers. Generally, after this amount of time, an OD 600 of 0.8 to 1.0 is expected. Place a sterile 96 well plate into the hood.
For each sample or control, prepare 3 wells with 199 microliters of sterile LB broth with ampicillin and arabinose. Next, pipette 1 microliter of the cell solution from the overnight broths into individual wells of the 96 well plate. Fill the plates with a breathable cover and then shake them for 18 to 20 hours at 37 degrees Celsius and 160 rpm.
The next day, use a plate reader to determine the OD 600, which should be approximately 0.5. Also, measure the G of P fluorescence at an excitation wavelength of 395 nanometers and at emission of 509 nanometers. Measure RFP fluorescence at an excitation wavelength of 575 nanometers and emission of 605 nanometers.
Using Excel, normalize for the variation in growth by dividing the florescence by the OD 600. After normalization, determine the relative terminator strength by taking the GFP terminator fluorescence over GFP control fluorescence and dividing it by the RFP terminator fluorescence over the RFP control fluorescence. Fix putative terminators T1 through 6 bio-informatically identified in the microbacteria phage bernal 13 were analyzed as described in this video.
These results show relative fluorescence. All results were normalized using cells containing uncut, unmodified PGR as a control. T3 was found to be a strong terminator.
All results represent individual colonies grown in triplicate and averaged for visual representation. Error bars represent deviations between averaged data points. To determine the best reference control for quantifying terminator strength, raw data using the original pGR plasmid containing no terminator, and pGR blue cloned with a sequence known not to be a terminator, were compared.
As can be seen here, both controls showed similar levels of GFP and RFP expression and more importantly, produced similar GFP to RFP ratios. All of these results represent individual colonies that were grown in triplicate and averaged for visual representation. Once mastered, this technique can take as little as 72 hours including shipping and ordering time.
It is important to note that pGR blue only works in e.coli. Additionally, since we're only looking at a small piece of the terminator, we really have no idea of other regulatory elements that may control transcription. To verify the results you achieve with this protocol using pGR blue, additional methods can be used like quantitative PCR.
Additionally, the pGR insert can be cut out and ligated into a plasmid of your choice.
