Investigating the Relaxase Behavior and Replication Functionality of the Mobilization Protein mobV in the Plasmid pBBR1

Mark Kathol Author

04/02/2021 Added

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This presentation will be looking understanding the mechanisms behind plasmid retention to make further genetic engineering research easier

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[00:00:00.000]Hello, my name is Mark Kathol.
[00:00:03.492]I am a graduate student here
[00:00:05.300]at the university of Nebraska-Lincoln.
[00:00:07.580]I work at the SS bio lab headed by Rajib Saha,
[00:00:11.570]at the Chemical and Biomolecular Engineering Department
[00:00:15.470]here at the university.
[00:00:17.060]I am here to present both mine and Cheryl Immethun's
[00:00:19.720]work on investigating the relaxase behavior
[00:00:22.360]and replication functionality
[00:00:24.260]of the mobilization protein mobV in the plasmid pBBR1.
[00:00:29.770]So what is a plasmid?
[00:00:31.520]What is plasmid retention
[00:00:32.850]and why should we care about it?
[00:00:34.610]A plasmid is a bit of extra chromosomal DNA,
[00:00:38.210]which is replicated independently of the main chromosome.
[00:00:42.750]Plasmid retention occurs when
[00:00:44.698]all daughter cells of a parent cell
[00:00:47.820]receive at least one copy of the plasmid.
[00:00:50.620]Plasmid loss occurs
[00:00:52.370]when not all daughters cells receive at least one copy,
[00:00:55.490]which can lead to heterogeneous cell cultures,
[00:00:57.920]which is what we do not want.
[00:00:59.620]We want to set our genetic edits to stay
[00:01:02.470]in all copies of the cell at all times.
[00:01:05.400]There are two main methods
[00:01:06.790]of genetic engineering these days
[00:01:09.670]and performing genetic edits.
[00:01:11.340]One is transformation, the others genome editing.
[00:01:14.390]Transformation can be done
[00:01:16.140]through methods such as electroporation or heat shock,
[00:01:19.140]where plasmids are inserted into a cell by punching holes
[00:01:23.070]in the cell membrane and having the cell membrane heal
[00:01:25.960]over once the plasmid is inside,
[00:01:28.130]and editing the main genome
[00:01:29.870]through methods such as CRISPR and TALEN.
[00:01:32.290]These methods are easier these days than they were before
[00:01:36.750]but transformation still is much easier than genome editing
[00:01:40.440]and plasmids and transformation are the workhorse
[00:01:44.340]of genetic engineering these days.
[00:01:48.020]However there are some problems
[00:01:49.530]when performing genetic engineering
[00:01:51.030]in non-model bacteria such as polyploidy
[00:01:54.790]where a cell can contain multiple copies of its main genome.
[00:01:59.900]Which means that our genetic edits can be lost
[00:02:02.680]through the same mechanism over here on the left.
[00:02:06.140]Another challenge is
[00:02:07.880]antibiotic resistance, antibiotics are commonly
[00:02:10.370]used to kill bacteria, which do not have the plasmid.
[00:02:14.030]And if those bacteria remain despite those,
[00:02:16.750]the presence of antibiotics, then our cultures
[00:02:20.490]can become heterogeneous quickly, which is bad.
[00:02:23.530]Because again
[00:02:24.363]we want only homogenous cultures to characterize.
[00:02:28.210]So how are we going about understanding plasmid retention?
[00:02:33.260]A couple years back, we were transforming plasmids
[00:02:35.930]into our non-model organism, R. palustris
[00:02:39.470]with the exception of this mobilization gene.
[00:02:43.370]When our plasmids did not contain this gene
[00:02:47.786]our plasmids were not being retained
[00:02:49.762]for a long period of,
[00:02:51.577]a mobilization protein is a protein which assists
[00:02:54.162]in the conjugation of genetic material between bacteria.
[00:02:58.495]The mob gene is what encodes this mobilization protein.
[00:03:02.604]Our mob gene is very similar
[00:03:04.354]has a very similar amino acid sequence
[00:03:06.370]to a mob gene that has been previously annotated
[00:03:08.759]which has been found in pMV158.
[00:03:12.169]We have inserted it into our plasmid
[00:03:14.392]the mob gene from PBBR1.
[00:03:18.290]And we are investigating whether
[00:03:19.676]or not our mob gene has a similar active site composition
[00:03:24.850]to the previously annotated mob gene.
[00:03:28.156]What we are doing to determine this is that we are knocking
[00:03:32.622]out specific amino acids within the active sites to see
[00:03:36.283]whether or not it will have an effect on plasmids.
[00:03:40.833]This is what the active site looks
[00:03:43.434]like in the previously annotated mob gene.
[00:03:46.414]It uses a series of histidines rather than tyrosines
[00:03:50.242]which are more commonly used by relaxases.
[00:03:57.004]Our hypothesis is that the mob gene
[00:03:59.494]in pBBR1 also contains these histidine amino acids
[00:04:04.266]within their active sites.
[00:04:06.843]We currently have one data set to support our hypothesis
[00:04:11.233]which is flow cytometry.
[00:04:13.166]Flow cytometry works by passing bacteria through a laser
[00:04:17.333]agitating the fluorescent protein within the plasmid
[00:04:20.483]and seeing how many cells still have the plasmid.
[00:04:25.508]The top left graph is an example of a negative control.
[00:04:29.046]It is a cell culture which contains no plasmid whatsoever.
[00:04:33.658]The top middle graph is an example
[00:04:35.768]of a cell culture that contains plasmids
[00:04:39.776]and some cells which do not.
[00:04:43.410]The remaining graphs here
[00:04:45.532]represent the edits that we have made to the model.
[00:04:49.584]This dividing line is what determines whether
[00:04:51.691]or not a cell is considered to have a plasmid.
[00:04:54.573]If it is on the left
[00:04:55.582]then the cell is considered not to have a plasmid.
[00:04:58.856]If it is on the right
[00:05:00.125]then the cell is considered to have the plasmid.
[00:05:03.187]As you can see here from the edits that we have made
[00:05:06.707]the proportion of cells
[00:05:08.799]on the right has decreased from our positive control
[00:05:12.143]thereby supporting the hypothesis that the mob gene
[00:05:19.838]that we are studying also contains these active sites.
[00:05:24.028]We will be collecting two additional data sets
[00:05:26.980]to support this hypothesis.
[00:05:29.678]One of these data sets will be a plasmid relaxation assay
[00:05:33.319]this will determine whether
[00:05:34.317]or not our edits are still de-supercoiling the plasmid
[00:05:38.749]helping the replication machinery attached to it
[00:05:42.056]and therefore make more copies.
[00:05:43.956]If a plasmid is being de-supercoiled by our edited protein
[00:05:47.825]then it will run slower than the supercoiled plasmid.
[00:05:55.031]This is only an example
[00:05:56.142]of a plasmid relaxation assay,
[00:05:58.179]the supercoiled plasmid would be on the bottom
[00:06:01.132]which would run faster in a gel than relaxed plasmids.
[00:06:07.217]The second data set we will collect is qPCR
[00:06:10.046]which will try to correlate the mob genes
[00:06:12.406]transcription rate with the plasmids replication protein,
[00:06:16.117]and see whether or not that is correlated
[00:06:19.876]to the increase of plasmid retention within the cell.
[00:06:23.977]This is the current state of our work
[00:06:25.376]and we are looking forward to collecting more data
[00:06:28.197]to understand the plasmid retention effects
[00:06:31.001]of the mobilization gene.
[00:06:33.900]This was supported by the Nebraska Public Power District
[00:06:35.707]through the Nebraska Center for Energy Sciences Research
[00:06:37.818]at the University of Nebraska Lincoln
[00:06:40.884]it is also supported through the NSF career award
[00:06:43.677]I would also like to acknoledge the rest of the
[00:06:46.298]Systems and Synthetic Biology Lab
[00:06:48.433]Headed by Dr. Rajib Saha
[00:06:50.337]I would also especially like to acknowledge Dr. Cheryl Immethun
[00:06:54.691]who has contributed massively to this work
[00:06:57.682]Thank you for watching this presentation

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Investigating the Relaxase Behavior and Replication Functionality of the Mobilization Protein mobV in the Plasmid pBBR1

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