Constructing a Plasmid

Grace Troupe, Presenter Author

10/11/2018 Added

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One way to put new genes in plants is by using the soil bacteria Agrobacterium. This process is started by putting your transgene into a plasmid so that the bacteria has the proper instructions for sending the gene into plant cells. This video walks through the general steps of putting a transgene into a plasmid.

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[00:00:06.150]In plant genetic engineering
[00:00:07.880]it's common to use a soil bacteria called agrobacterium
[00:00:12.120]to deliver genes into plant cells.
[00:00:15.180]This technique takes advantage
[00:00:16.880]of agrobacterium's natural ability
[00:00:19.140]to insert genes into plants.
[00:00:21.640]In this video, we'll look at how this bacteria
[00:00:24.100]is given a specific gene to deliver.
[00:00:27.330]This is done using a circle of DNA called a plasmid,
[00:00:30.900]and this rotating DNA represents a plasmid.
[00:00:36.950]In nature agrobacterium usually carries a plasmid
[00:00:40.300]that contains genes which cause plant cells
[00:00:42.820]to make food for the bacteria.
[00:00:45.150]These genes can be cut out of the plasmid
[00:00:47.860]and replaced with the gene that you want to deliver.
[00:00:51.470]By putting your gene in the same spot,
[00:00:53.870]the bacteria will know it should be transferred.
[00:00:56.880]This image shows that a plasmid is cut using a protein
[00:01:01.160]and now it's ready to have a transgene added.
[00:01:05.100]The overhangs where the plasmid was cut
[00:01:07.540]must match with the gene so it can become part
[00:01:10.460]of the plasmid circle.
[00:01:12.500]These overhangs are called sticky ends
[00:01:14.900]because the matching nucleotide sequence
[00:01:17.760]of the gene and the plasmid are attracted
[00:01:20.170]to each other, thus making them sticky.
[00:01:24.220]The cut plasmids are now in the tube on the left.
[00:01:27.450]These are mixed with the transgenes
[00:01:29.410]in the tube on the right.
[00:01:30.910]They have matching sticky ends so that the gene
[00:01:33.600]can become a part of the plasmid.
[00:01:36.090]Since these are small fragments of DNA,
[00:01:38.450]they can't actually be seen with the naked eye.
[00:01:48.134]If you could see what was going on,
[00:01:50.380]this would be about how it would look.
[00:01:52.420]The fragments are floating around in solution.
[00:01:55.420]Then when they encounter one another,
[00:01:57.760]the attraction of the sticky ends
[00:01:59.500]will cause them to attach.
[00:02:02.050]So here it is in action.
[00:02:07.740]The attraction of the nucleotides hold the fragments
[00:02:10.370]together, but then some proteins act like tape
[00:02:14.500]and they come seal the pieces together
[00:02:16.350]to be sure they won't come apart.
[00:02:23.390]Finally, we have a plasmid with the gene
[00:02:25.680]we want to deliver.
[00:02:27.370]The gene is flashing on the side
[00:02:29.180]to show the portion of the plasmid that we added.
[00:02:32.750]The next step in this process would be
[00:02:35.000]to give the plasmid to a disarmed agrobacterium.
[00:02:38.230]Then this bacteria can deliver the gene to plant cells.
[00:02:42.720]And this is part of the process of plant transformation.

Constructing a Plasmid

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Constructing a Plasmid

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