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General outline of the method:

1. Polymerase Chain Reaction (PCR) to obtain DNA fragments
2. Gel electrophoresis to select the correct fragments
3. DNA purification for plasmid assembly
4. Gibson Assembly
5. E. coli transformation and plating
6. Colony analysis and selection by colony PCR
7. Miniprep and sequencing

PCR

In this step we will amplify the fragments required for our plasmids with Phusion DNA polymerase. PCR will amplify a fragment from a DNA template, with the help of short DNA sequences complementary to the template that demarcate the ends of the fragment. In our case, the template will be a similar plasmid with RFP-LTI in place of our GOI.

!!Put in picture of the plasmid !!

The plasmid backbone will be split into 3 pieces, as it is quicker and less error prone to PCR short fragments (<5kb). The fragments will be amplified with the following pairs of single stranded DNA primers (length of the fragments in brackets):

1. nosT_F and P2_B (2137bp)
2. P2_F and P1_B (2501bp)
3. P1_F and 35s_B (3000bp)

A fourth fragment will be our GOI All the fragments are designed to overlap with each other by 20-40 bp for the subsequent Gibson assembly reaction.

PCR Protocol

1. Add primer and template DNA to PCR tubes (label them)
2. Create the phusion mix in a 1.5 ml eppendorf (note this is slightly more mix that is required (for 4 tubes), since we want to ensure that we will have enough):
   • HPLC H20 162.5 µl
   • 5x HF buffer 50 µl
   • 10mM dNTPs 5 µl
   • Phusion polymerase 2.5 µl
3. Add 44 µl of the phusion mix into each tube containing DNA (shake or centrifuge them first to ensure that the DNA solution is in the bottom of the tube).
4. Place into a PCR machine and set the phusion protocol running (this is directly from the NEB phusion protocol):
   • 30 sec of 98°C (initial denaturation)
   • 30 cycles of:
     • 10 sec of 98°C (denaturation)
     • 20 sec of 58°C (annealing)
     • 2:00 mins of 72°C (extension)
   • 5 mins of 72°C (final extension)
   • Hold at 4°C

This reaction will take roughly 2 hours, and can be kept on hold at 4°C without problems for many hours after completion. Next, we will proceed to the gel electrophoresis step.

Gel Electrophoresis

The PCR tubes will now contain our fragments, as well as template and primer DNA that will interfere with the subsequent Gibson assembly reaction and transformation. Therefore we will need to extract and purify the right DNA fragments. To do this, we will run the mixture through an agarose gel by applying a voltage, which will cause the negatively charged DNA to migrate. Larger fragments will migrate slower, and thus DNA molecules will be separated by size. It will also allow us to confirm that we have the correctly sized DNA. Firstly, we should make a 100 ml 1% (w/v) agarose TAE gel:

1. Weigh out 1g Ultrapure agarose, and add it to a glass flask
2. Add 100 ml 1xTAE
3. Microwave for 2:30 mins, swirling the flask after 1:30 mins
4. Place in 55°C hybridizer to cool for 20-30 mins (when the gel is too hot, the mould expands and the molten gel leaks out)
5. Pour into two 50 ml falcon tubes
6. Add 5 µl of Sybr Safe DNA dye to each falcon (do this in a dedicated gel area – DNA dyes are generally not good for health)
7. Pour out both falcons into the gel mould with 2 of the 8 toothed combs and leave to set for 30 mins
8. Once the PCR is finished, add 12.5 µl of 5x loading dye to each PCR tube
9. Then, remove the combs and walls from the set gel, and place into the gel tank containing enough TAE to cover the gel
10. Load the gel lanes – lane 1 of each level with Hyperladder 1kb, and the remaining lanes with the results of your PCR
11. Run at 100V for 40 mins