Team:Cambridge-JIC/Protocol

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.

Plasmid which carries the gene of interest

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):

nosT_F and P2_B (2137bp)
P2_F and P1_B (2501bp)
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

Add primer and template DNA to PCR tubes (label them)
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
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).
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:

Weigh out 1g Ultrapure agarose, and add it to a glass flask
Add 100 ml 1xTAE
Microwave for 2:30 mins, swirling the flask after 1:30 mins
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)
Pour into two 50 ml falcon tubes
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)
Pour out both falcons into the gel mould with 2 of the 8 toothed combs and leave to set for 30 mins
Once the PCR is finished, add 12.5 µl of 5x loading dye to each PCR tube
Then, remove the combs and walls from the set gel, and place into the gel tank containing enough TAE to cover the gel
Load the gel lanes – lane 1 of each level with Hyperladder 1kb, and the remaining lanes with the results of your PCR
Run at 100V for 40 mins

DNA purification

We will now recover our fragments from the gel. Firstly, prepare enough appropriately labelled eppendorfs and a gel cutting tip for each tube. Once the gel is finished, remove it from the gel tank and take it to the dark room on the first floor in a container along with your tubes, gel cutting tips and a P1000. Under the blue light illuminator, find the appropriately sized DNA band and extract it using the pipette, placing the gel fragment into the appropriate tube. Afterwards, take a picture of the gel in the imaging box (The imaging box uses UV light, which can damage DNA, so do this after you cut out your band).

Once the gel fragment is in your tube, take it back to the lab and purify the DNA using the Qiagen Minelute kit, using the protocol provided in the kit.

Gibson Assembly

We can now combine our DNA fragments into the final circular plasmid. The Gibson assembly reaction is an isothermal one-pot reaction containing 3 enzymes that will convert the linear double stranded DNA fragments from the PCR into circular DNA. (For more information see the synbio.org guide, or Gibson et al., Nature Methods, 2009)

Pipette 0.5 µl of each DNA fragment into a PCR tube, for a total volume of 2 µl (make sure all the liquid is in the bottom of the tube)
Start the PCR machine running a Gibson protocol (50°C for 1 hour, then hold at 4°C)
Add 6 µl of 1.33x Gibson Master Mix to the tube
Place immediately into a PCR machine

Note that it is important to place the tube at 50°C very quickly after adding the master mix. If you wait too long (more than 20-30 secs), the exonuclease enzyme (which does not work well at 50°C) will degrade too much of the DNA, lowering the efficiency of the reaction significantly

E. coli Transformation

Now that we have our plasmids, we will transform it into E. coli for replication.

Get a tube of 50 µl E. coli chemically competent cells from the -80°C freezer, and place it on ice
Once the cells have thawed, place the whole Gibson reaction into the competent cells tube, and return the mixture to ice for 15-20 minutes
Place the mixture into a 42°C water bath for 1 minute
Return the tube to the ice for another minute
Add 250 µl of SOC
Place into a 37°C shaking incubator for 1 hour for recovery, giving the cells time to express the antibiotic resistance. Also place a 9mm petri dish containing LB agar and the appropriate antibiotic (kanamycin 50 in our case) in the incubator with the cells to warm up.
Plate the entire 300 µl onto LB agar plates, spreading the liquid over the plate with the L-shaped spreader
Incubate the plate overnight at 37°C

Note that the competent cells are very fragile, and must be kept on ice before the heat shock step. Also, do not to touch the bottom of the competent cell tubes with your hands, as this may warm them too much. Also, when adding the results of the Gibson reaction, do not pipette the cells up and down.

Colony PCR

Now that we (hopefully) have colonies containing plasmids, we need to choose a colony to grow to obtain the correct plasmid for further transformations into Agrobacterium and Marchantia. Misassembly is possible with Gibson, particularly if there are repeated sequences present or if the plasmid is somewhat toxic to the bacteria (which should not be a problem in our case).

We will now verify that the plasmid is correctly assembled by PCR. We will amplify a fragment across two Gibson junctions, to confirm that our insert has been correctly incorporated. To do this, we will use the 35s_seq and nosT_Bseq primers, which bind as shown on the diagram below. We can also use these primers for sequencing later.

!!Put in picture of the plasmid segment !!

Colony PCR Protocol:

Identify 8 colonies to screen, and label them (scale the reaction down if you have fewer than 8 colonies)
Make a mix with the phusion and primers in an eppendorf tube:
- HPLC H20 134 µl
- 5x HF buffer 40 µl
- 10mM dNTP 4 µl
- 10µM 35s_seq primer 10 µl
- 10µM nosT_Bseq primer 10 µl
- Phusion polymerase 2 µl
Prepare 9 small PCR tubes, and pipette 20 µl of the phusion mixture into each tube
With a small tip, touch a colony, and mix into the appropriate PCR tube. Note: do not pick up too much bacteria, as it will disrupt the PCR, a very small amount of DNA can act as a template 10
Onto the last PCR tube, place 1 µl of the 35s_RLTI template DNA to act as a positive control
Place into a PCR machine and run a Phusion protocol, with an additional lysing step:
- 6 mins of 98°C (lysing and denaturation)
- 30 cycles of:
  - 10 sec of 98°C (denaturation)
  - 20 sec of 58°C (annealing)
  - 1:00 mins of 72°C (extension)
- 5 mins of 72°C (final extension)
- Hold at 4°C
Prepare a gel whilst you wait (1% agarose (w/v) in TAE = 1g of UltraPure agarose in 100 ml of TAE), using the thinner toothed combs in the mould
Place the PCR products into the gel lanes (with 5 µl of loading dye), as well as Hyperladder 1kb in each row
Run for 40 mins at 100V
Image the gel

The correct band size depends on the construct is being built, but they should be around 1-1.2 kb. Select a colony with a correct band, and place with an inoculation loop into an unskirted falcon containing 15 ml of LB + kanamycin 50µg/ml. Leave overnight in a 37°C shaking incubator.

Miniprep

We will now harvest the plasmids from the E. coli for further use. We will do this using the Qiaprep kit, so follow the protocol provided in the kit. Colony PCR can only tell us if the fragment is the correct length, which is indicative of a correct assembly (and only in the junctions we are PCRing over). Once we have our plasmids, we should sequence them to confirm that their sequences are correct. To do this, obtain an account with Source Biosciences, and send them the appropriate amount of DNA and primers for Sanger sequencing.

After receiving the sequencing data, we will (hopefully) have a newly constructed DNA plasmid with the sequence we wanted. Congratulations, you have now built a new plasmid!

The protocol for Marchantia:

Day 1 (need primers & template DNA)

PCR (3 hours)
Gel purification (2 hours)
Gibson (1.5 hours)
E. coli transformation (1.5 hours then overnight)

Day 2 & 3

Select E. coli
50ml culture
Plasmid extraction
Marchantia spore sterilisation & plating

Day 4

Agrobacterium transformation

Day 7

Pick Agrobacterium and grow overnight

Day 8

Transform Marchantia

Day 10

Plate Marchantia transformants

Day 13+

Results