TITLE: Library cloning
AUTHORS: Raining Wang, Melinda Wheelock

[DESCRIPTION]
This is the protocol for inserting the library gene of interest into the pre-barcoded vector created in the previous protocol (Barcoded Vector Cloning). 

[BEFORE_START]
Ensure there are enough maxi-prep kits available to use.

[STEPS]
SECTION: Predigest the barcoded backbone
1. Set up a 50 µL reaction as outlined below:
 
 
 
SECTION: Predigest the barcoded backbone
2. Set the thermal cycler for the program below:
 
 
 
 

SECTION: Predigest the barcoded backbone
3. Run all digested samples on a 0.5% TBE gel to ensure complete digestion of the backbone.

Also run1 µg of original starting template as a visual reference for the supercoiled plasmid.

SECTION: Predigest the barcoded backbone
4. Gel extract the linearized product.

SECTION: Predigest the barcoded backbone
5. Use the Clean & Concentrator kit on the gel extraction product.

To avoid issues with freeze-thaws, aliquot the cleaned, linearized product in small volumes.

SECTION: Golden gate assembly of variant library inserts
6. 
 

Set up reaction as outlined below:
SECTION: Golden gate assembly of variant library inserts
6.1. 
 

SECTION: Golden gate assembly of variant library inserts
6.2. Here is a ligation calculator by NEB to use if you don't have a formula set up.

SECTION: Golden gate assembly of variant library inserts
6.3. Here is NEB's general protocol for GG with SapI.

SECTION: Golden gate assembly of variant library inserts
6.4. Neg Ctl: Using the vector with no insert.

 
 
 
SECTION: Golden gate assembly of variant library inserts
7. Set a thermocycler for the following program:

Spike in 1ul of SapI every hour.
 
 
SECTION: Golden gate assembly of variant library inserts
8. Set up additional digestion reaction as follows:
 
 
 
SECTION: Golden gate assembly of variant library inserts
9. Incubate at 37 °C for at least 120 min, or 120 min. 

Spike in SapI every hour.

SECTION: Golden gate assembly of variant library inserts
10. Purify the product with the Zymo Clean & Concentrator kit.

Pool 2 sample reactions (40 µL) for 4 columns.

SECTION: Golden gate assembly of variant library inserts
11. Measure the final eluate with Nanodrop to look for a normal purity curve.

260/280 ratio should be:

SECTION: Golden gate assembly of variant library inserts
12. Quantify the product concentration with Qubit BR assay.	

The final eluate should be >5ng/uL for the samples and very low for the negative control.

SECTION: Transformation with electro-competent cells
13. Prepare cells for transformation
SECTION: Transformation with electro-competent cells
14. Prepare for recovery

SECTION: Transformation with electro-competent cells
15. Electroporation

SECTION: Transformation with electro-competent cells
16. Sample recovery

SECTION: Estimating barcode coverage for bottlenecking
18. Count colonies on dilution plates to calculate the approximate variant to barcode coverage

For examples:
Library X : 205 x 22 variant changes = 4510 aa variants. We want to have to pick the flask that produces between 45,100 -135,300 colonies. This indicates between 10-30x coverage over the variants.

 

 
 
SECTION: Transformation with electro-competent cells
13.1. Warm SOC to 37 °C.
SECTION: Transformation with electro-competent cells
13.2. Chill empty cuvettes on ice for 15 min.
SECTION: Transformation with electro-competent cells
13.3. Thaw NEB electrocompetent cells (100 uL/tube) on ice for approximately 5 min.
SECTION: Transformation with electro-competent cells
13.4. To chilled cuvettes, add 26 µL of thawed cells.

Pipette to first stop only - do not create air bubbles as this will cause the electroporation to fail.
Can use long gel-loading tips to do this more easily.
SECTION: Transformation with electro-competent cells
13.5. Add 1 µL-2 µLof cleaned DNA from golden gate reaction and controls to the corresponding cuvette containing cells.

Pipette to first stop only - do not create air bubbles.
Do not add more than 3 uL of plasmid.
SECTION: Transformation with electro-competent cells
13.7. Incubate on icefor 30 min.
SECTION: Transformation with electro-competent cells
13.6. Flick the cuvette gently, then lightly tap on the bench. Place the cuvette back on ice.
SECTION: Transformation with electro-competent cells
14.1. While the cells are incubating on ice, prepare glass recovery tubes with 900 µL SOC each, and warm to 37 °C.
SECTION: Transformation with electro-competent cells
14.2. Set the MicroPulser Electroporator to Manual for 2.0 ms.

The electroporator is automatically set for 6 kV.
SECTION: Transformation with electro-competent cells
14.3. Prepare six overnight growth Erlenmeyer flasks with 300 mLof LB + 30 µL carbenicillin for each sample.

Use 500 mL flasks for enough air circulation.
SECTION: Transformation with electro-competent cells
14.4. Label the LB + ampicillin dilution plates for each sample. Place in 37 °C after labeling to pre-warm.

For Negative Golden Gate control, need 2 dilution plates
For library and pUC19, need 3 dilution plates each.
SECTION: Transformation with electro-competent cells
15.1. Starting with the negative control cuvette, wipe the outside of the cuvette dry to remove condensation and ice.
SECTION: Transformation with electro-competent cells
15.2. Place the cuvette in the electroporator, ensuring the metal on the cuvette makes contact with the electrodes, and press the Pulse button.

If the electroporator makes a loud noise and displays “Arc” on the digital readout, the sample has failed and is no longer good.
SECTION: Transformation with electro-competent cells
15.3. Remove the cuvette from the electroporator and place back on ice.
SECTION: Transformation with electro-competent cells
15.4. Repeat with all cuvettes prepared.
SECTION: Transformation with electro-competent cells
16.1. Remove the glass recovery tubes containing the SOC from 37 °C.
SECTION: Transformation with electro-competent cells
16.2. Transfer the cells from each cuvette to the glass recovery tubes.
SECTION: Transformation with electro-competent cells
16.3. From the warmed bottle of SOC, add 100 µL to wash the electroporation cuvette, then transfer from the cuvette to the corresponding glass recovery tube. Repeat this for all samples.

Long gel-loading tips may be helpful for sample recovery from the cuvettes.
SECTION: Transformation with electro-competent cells
16.4. Place the glass recovery tubes into the shaking incubator for 60 min at 37 °C.
SECTION: Transformation with electro-competent cells
17. Prepare dilution tubes and plates
SECTION: Transformation with electro-competent cells
17.1. As the cells are recovering, prepare microcentrifuge tubes for serial dilution of the library and controls. We recommend to start with a bottleneck scheme by dividing the 1ml recovery culture to 6 portions: 500 µL, 250 µL, 125 µL, 2x 50 µL, and 25 µL. 

For each portion, prepare 4 tubes (D0-D3) with the listed volume of LB. Label three plates (D1-D3) for each bottleneck portion (total of 18 plates). 
 
 
SECTION: Transformation with electro-competent cells
17.2. For Puc, prepare two tubes and plates as the following Negative control and H2O. 
 
 
SECTION: Transformation with electro-competent cells
17.3. For Negative control and H2O, plate 100 mL from the 1 mLrecovery culture to the respective plates.
SECTION: Transformation with electro-competent cells
17.4. 
 
After the 1 hour recovery, add the appropriate portion(as below) from the 1 mL culture to the respective 500-mL flask based on the bottlenecking scheme.
Place all 500 flasks in the 37 °C shaking incubator for 3 min to ensure it is well mixed. No need to prepare overnight growth flasks for Puc19, negative control, and H2O controls. 
 
 
SECTION: Transformation with electro-competent cells
17.5. Mix each dilution well. Plate each dilution according to the dilution scheme above.  Incubate 3 min at 37 °C. 
SECTION: Transformation with electro-competent cells
17.6. Incubate 3 min growth flasks in 30 °C for 840 min-960 min.