The following videos details a procedure for using the C-Brick standard to assemble DNA parts using CRISPR-associated protein, Cpf1. This method can further the development of DNA assembly in the field of synthetic biology. Such as by facilitating the construction of complicated genetic circuits.
The main advantage of this method is that is it sequence independent and allows for iterative assembly of DNA constructs. We first had the idea for this method when we noticed that Cpf1 was guided by single siRNA and catalyctic DNA being stacked away. Therefore, generating DKNs.
Demonstrating the procedure will be Shi-Yuan Li, a graduate student from my laboratory. To repair the crRNA templates, re-suspend the individual oligonucleotides in RNase free water to a concentration of 10 micromolar. To a 0.2 milliliter PCR tube, add top strand oligonucleotide, bottom strand oligonucleotide, and 10x annealing buffer as detailed in the accompanying document.
Ensure that the total volume is 50 microliters. This table shows six different bottom strand oligonucleotides, each of which should be individually paired with the top strand, T7-F. The lowercase letters represent the sequence to be transcribed into the guide sequence of crRNA.
Place the PCR tube into a thermocycler. Run the annealing program with an initial denitration at 95 degrees celsius for five minutes, and then a cool down from 95 to 20 degrees celsius with a one degree celsius decrease per minute. Following annealing, immediately perform transcription by combining the annealed product with RNase free water, T-7 transcription buffer, NTP mixture, recombinant RNase inhibitor, and T-7 RNase polymerase in a 1.5 milliliter microcentrifuge tube.
Incubate the reaction in a water bath at 37 degrees celsius overnight. The next day, use an RNA clean-up and concentration kit to purify the transcribed RNA. Use a UV-Vis spectrophotometer to quantify the RNA.
Then, dilute the RNA to a concentration of 10 micromolar. If the samples are not used immediately, store them at 80 degrees celsius. Construction of C-Brick parts can be accomplished by several methods.
In this section of the video, three chromoprotein cassettes, cjBlue, eforRed, and amilGFP will be assembled using the seamless assembly method. Begin by designing and ordering the oligonucleotides for PCR amplification of the chromoprotein cassettes. When the oligonucleotides arrive, re-suspend them in ultrapure water to a concentration of 10 micromolar.
Next, to set up the PCR reactions, place 0.2 milliliter PCR tubes on ice. Add ultrapure water, two XPCR buffer, DNTPs, forward and reverse primers, chromoprotein template, and high fidelity DNA polymerase. Place the tubes directly into the thermocycler and run the program.
Use the samples immediately, or freeze and store them at 20 degrees celsius. Following amplification, purify the PCR products using a kit. Next, to digest the C-Brick standard vector with BamH1, add ultrapure water, plasmid, 10x buffer, and BamH1 to the tube.
Incubate for up to two hours at 37 degrees celsius in a water bath. Purify the linearized vector using a gel cleanup kit. Following the cleanup, combine linearized vector, the PCR product, 5x seamless assembly buffer, and seamless assembly enzyme from a seamless assembly kit in a 1.5 milliliter microcentrifuge tube.
Ensure that the total volume is 10 microliters. Place this in a 37 degree celsius waterbath for 30 minutes. Following the incubation, use the samples immediately or freeze them on ice and store them at 20 degrees celsius.
To 90 microliters of e coli DH10B, add 10 microliters of the construct. Perform a chemical transformation. Then, spread the transformed cells on an LB plate, and incubate at 37 degrees celsius.
The next day, select several clones to culture in five milliliter liquid LB tubes and incubate them at 37 degrees celsius on a shaker at 220 rpm overnight. Extract the plasmid using a plasmid preparation kit. Then, identify the correct clones by Sanger sequencing.
Know that the construction of C-Brick paths can also be accomplished by the reactive PCI application, or by restriction in the immediate digestion of T4 DNA ligase medium to ligation. Next, to digest the C-Brick vector with Cpf1, combine RNase free water, 10x Cpf1 buffer, the plasmid, recombinant RNase inhibitor, and Cpf1 in a 1.5 milliliter microcentrifuge tube. To insert a foreign DNA fragment into the T1 and T2 sites, add one microliter of crRNA T2, and incubate the tube in a water bath at 37 degrees celsius for 30 minutes.
Then, keeping the tube in the water bath, add one microliter of crRNA T1, one microliter of Cpf1, and incubate for another 30 minutes. The crRNA T2 is added first, because the two recognition sites are closer on the vector DNA. Cpf1 binding on the T1 site may influence the T2 site's bedding.
Also, if the T2'site was replacing the T2 site, the T2'site would be disrupted if the crRNA T1 was added first. Add one microliter of thermosensitive alkaline phosphatase and incubate the tube in a 37 degree celsius water bath for another hour. Perform agarose gel electrophoresis and purify the vector using a gel cleanup system kit.
Use the samples immediately, or freeze and store them at 20 degrees celsius. To digest the DNA fragment of interest with Cpf1, combine RNase free water, 10x Cpf1 buffer, plasmid, recombinant RNase inhibitor, and Cpf1 in a 1.5 milliliter microcentrifuge tube. To insert a foreign DNA fragment into the T1 and T2 sites, add one microliter of crRNA T1 and one microliter of crRNA T3 to the tube and incubate it in a water bath at 37 degrees celsius for two hours.
After the incubation, perform agarose gel electrophoresis and purify the fragment using a gel cleanup kit. Use the samples immediately, or freeze and store them at 20 degrees celsius. Next, to ligate the foreign DNA fragment in C-Brick vector, combine the linearized vector and DNA fragment in a 1.5 milliliter microcentrifuge tube.
Add 10x T4 DNA Ligase Buffer, and T4 DNA Ligase. Then, incubate for two hours at 22 degrees celsius. Use the samples immediately, or freeze and store them at 20 degrees celsius.
Add 10 microliters of the ligation products to e coli DH10B competent cells, and perform a chemical transformation. Then, spread the transformed e coli onto LB plates, and incubate them at 37 degrees celsius. The next day, add several clones to 5 milliliter liquid LB tubes and incubate them overnight on a shaker at 220 rpm and 37 degrees celsius.
Extract the plasmid using a plasmid preparation kit. Then, identify the correct clones by Sanger sequencing. Perform a new round of C-Brick assembly.
Using the sample, so called crack clones can be used as a new C-Brick vector or DNA piece for further C-Brick iterative assembly. Three chromoprotein cassettes, cjBlue, eforRed, and amilGFP, were assembled as described in this video. Three chromoproteins were expressed in e coli grown on a plate.
Negative control bacteria, with no chromoproteins, are shown on the fourth plate. Subsequently, two color expression cassettes were further assembled with the C-Brick standard to create more colors. Three chromoproteins and three assembled dual chromoproteins were expressed in e coli grown in liquid LB medium.
From one to six, the constructs expressed are eforRed, one, amilGFP, two, cjBlue, three, amilGFP plus eforRed, four, eforRed plus cjBlue, five, and amilGFP plus cjBlue, six. The colorful bacteria on a single plate, or in a single microcentrifuge tube, were cultured from a single sequence verified clone. Once mastered, this technique can be done in one week, if it is performed properly.
While attempting this procedure, it is important to remember that all materials should be RNase free, for siRNA preparation and Cpf1 clearancing. I still recommend, this tactic has helped as a way for researchers in the field of synthetic biology to build a genetic showcase of metabolic pathways. After watching this video, you should have a good understanding of how to assemble a DNA path using C-Brick standard.
