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2. Library Cloning
Upon receiving the oligo pool from TWIST (which typically takes ~1-2 weeks) or IDT, it is necessary to amplify the oligo pool with a small number of PCR cycles to increase the amount of DNA.
In first part of the protocol, we discussed the importance of pooling small groups of mutated sequences together; typically 3-5 genes for each primer pair. This grouping saves time in this part of the protocol, as it eliminates the need to perform a separate PCR reaction for each gene (and its 1200-1500 mutated versions).
The TWIST website includes instructions on oligo pool amplification. We recommend that you familiarize yourself with that page before proceeding.
An initial amplification of the aliquoted oligo library ensures that we have ample DNA for cloning of the library into plasmids, followed by transformation into E. coli or another organism of interest. This part of the protocol assumes that you have divided the oligo library into smaller aliquots, at a concentration of 10 nanograms per microliter. For long-term storage, these DNA libraries should kept at -80C.
When you are ready to work with the DNA, follow the TWIST guidelines to amplify the oligo library.
Remove an aliquot (we recommend 5 microliters per aliquot) of the oligo pool, and set up one 50 microliter PCR reaction per primer pair. If you have 5 primer pairs (corresponding to up to 25 gene libraries), this would be 5 PCR reactions.
For each PCR reaction:
| reagent | concentration | volume |
|---|---|---|
| DNA (oligo pool) | 10ng/microliter | 1 microliter |
| Q5 polymerase mix | 2x stock | 25 microliters |
| Primer Fwd | 10 micromolar | 2.5 microliters |
| Primer Rev | 10 micromolar | 2.5 microliters |
| Water | N/A | 19 microliters |
Mix these reagents carefully, while keeping everything on ice throughout the experiment. The 'Primer Fwd' and 'Primer Rev' will differ based on the 20-nucleotide sequences flanking each group within the oligo pool (e.g. Kosuri-101 is a primer set that is distinct from Kosuri-102. Most of these primer pairs are already available in the Phillips laboratory, and are stored in the -20°C freezer).
Use the following thermocycler settings with 12 cycles.
| cycles | temperature | time |
|---|---|---|
| 1 | 98°C | 30 seconds |
| 12 | 98°C | 10 seconds |
| 12 | 64°C (anneal) | 30 seconds |
| 12 | 72°C (extend) | 30 seconds |
| 1 | 72°C | 120 seconds |
For more information on primer annealing temperatures (and a very user-friendly annealing temperature calculator), visit the NEB website's Tm calculator. For more information on the thermocycler conditions for Q5 polymerase, see the NEB website.
Given the low concentration of DNA involved, even after 12 cycles of amplification, loss of material is a major concern at this stage. Therefore, we recommend that you use column-based DNA purification methods to "clean" the amplified DNA. The Zymo DNA Clean & Concentrator-5 works well for this purpose.
After performing the PCR purification according to manufacturer's protocol, NanoDrop the eluted DNA and record the concentration and purity. Store away DNA at 4°C (if you will use in the next 24 hours) or at -20°C for longer term storage.
After initial amplification, it is time to add barcodes to each of the gene groups. Barcoding DNA, as a process, involves adding random, unique, 20-nucleotide "barcodes" to a DNA sequence of interest. Adding [DISCUSS qPCR HERE] While TWIST recommends 12-14 cycles of amplification for oligo libraries of 151-200 bases in length, the "ideal" number of cycles can only be accurately determined by quantitative-PCR (qPCR). qPCR serves as an important control because it ensures that the DNA for each group (with orthogonal primer pairs) is being amplified at relatively even levels. qPCR will also help you select the precise number of cycles to perform.
13 cycles qPCR walkthrough What is annealing temp and extension time? Which polymerase? Read about LUNA and other options, what does Bill use?
After amplifying the oligo libraries, perform a gel extraction by adding 10 microliters of 6x NEB DNA dye to each 50 microliter PCR reaction. Load the full volumes on a thick, 2% agarose gel. Electrophoresis for 45 minutes at 120V. Use a scalpel to remove the DNA band corresponding to the amplified oligo libraries. Perform a gel extraction using one of many commercially-available kits. We have previously obtained good results with the Zymoclean Gel DNA Recovery Kit.
The barcode was inserted 110 base pairs from the 5’ end of the mRNA, containing 45 base pairs from the targeted regulatory region, 64 base pairs containing primer sites used in the construction of the plasmid, and 11 base pairs containing a three frame stop codon. All the sequences are listed in Supplementary Table 1. Following the barcode there is an RBS and a GFP coding region.
Add Benchling files //
Mutated promoters were PCR amplified and inserted by Gibson assembly into the plasmid backbone of pJK14 (SC101 origin).
Add Benchling files //
[ADD NOTES OR IDEAS ABOUT GENOMIC INTEGRATION, USE OF pLIB]
Constructs were electroporated into E. coli K-12 MG1655 [54].
Cells were grown to an optical density of 0.3 and RNA was then stabilized using Qiagen RNA Protect (Qiagen, Hilden, Germany). Lysis was performed using lysozyme (Sigma Aldrich, Saint Louis, MO) and RNA was isolated using the Qiagen RNA Mini Kit. Reverse transcription was performed using Superscript IV (Invitrogen, Carlsbad, CA) and a specific primer for the labeled mRNA.
The growth conditions studied in this study were inspired by [1] and include differing carbon sources such as growth in M9 with 0.5% Glucose, M9 with acetate (0.5%), M9 with arabinose (0.5%), M9 with Xylose (0.5%) and arabinose (0.5%), M9 with succinate (0.5%), M9 with fumarate (0.5%), M9 with Trehalose (0.5%), and LB. In each case cell harvesting was done at an OD of 0.3. These growth conditions were chosen so as to span a wide range of growth rates, as well as to illuminate any carbon source specific regulators. We also used several stress conditions such as heat shock, where cells were grown in M9 and were subjected to a heat shock of 42 degrees for 5 minutes before harvesting RNA. We grew in low oxygen conditions. Cells were grown in LB in a container with minimal oxygen, although some will be present as no anaerobic chamber was used. This level of oxygen stress was still sufficient to activate FNR binding, and so activated the anaerobic metabolism. We also grew cells in M9 with Glucose and 5mM sodium salycilate. Growth with zinc was preformed at a concentration of 5mM ZnCl2 and growth with iron was performed by first growing cells to an OD of 0.3 and then adding FeCL2 to a concentration of 5mM and harvesting RNA after 10 minutes. Growth without cAMP was accomplished by the use of the JK10 strain which does not maintain its cAMP levels.
Details here.