Plasmid Construction

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Introduction

Plasmid construction is a key technique employed in genetic manipulation of bacteria. This meeting will provide a general understanding of plasmid structure and discuss the key steps in constructing a plasmid for genetic engineering purposes.

Microbial Genetics

Microbial genetics is a field of science that focuses on the expression systems of microorganisms. The study of biochemical pathways of microbes provides insights to gene recombination and evolution processes. Microbes are often used in genetic engineering as they have short generation time, easy to culture, and often occur in high abundance.

Genome vs Plasmids

Figure 1: Bacterium genome and plasmid. [1]

Genome refers to the complete set of genetic material in the organism. It contains all the genes necessary for function, therefore it is very long and complex. There is only one copy of the genome in an organism and it only replicates during cell division.


Plasmids are small circular DNA fragments that exist in the cytoplasm of bacteria or protozoa. There could be multiple copies of the same plasmid, and a bacterium could host multiple different plasmids. They could replicate independently and are passed on through replication or horizontal gene transfer.


Figure 2: E.coli genome. [2]
Figure 3: A plasmid. [3]

Function of Plasmids


Plasmids do occur in microbes naturally. They often carry genes that are advantageous to the host, like antibiotic resistance, degradation of heavy metal, or toxin production. The plasmids could either originate from the host or acquired from other microbes through horizontal gene transfer.


For the purpose of genetic engineering, plasmids are often used in labs to introduce foreign genetic material into a cell to produce recombinant DNA or proteins.

Components of a Plasmid


Origin of Replication (ORIs)

This is the location where DNA replication begins. Replication of plasmid is needed during cell division, or when a copy of the plasmid is made for horizontal gene transfer. There are many types of ORIs, and they determine the control of replication. With relaxed control, the plasmids can replicate independent of the host’s initiation process [4]. On the other hand, when a plasmid is under stringent control, it can only replicate as the host begins DNA replication [4].


Promoter Region

This region is where proteins required for transcription attach and drive the expression of the target gene. Different types of promoters determine the cell types in which the gene is expressed, and the activators/ inhibitors can alter the amount of recombinant protein obtained.


Multiple cloning site (MCS)

The MCS, also called the polylinker region, is a region on a plasmid that contains multiple cut sites for restriction enzymes. Restriction enzymes are enzymes that cleave DNA at specific sequences, which would allow foreign DNA to be inserted into the plasmid. The MCS often occur downstream of the promoter, and it is a special feature of engineered plasmid.


Selection Marker

This is a gene in the plasmid that is used for identifying successfully engineered bacteria. It is usually an antibiotic resistance gene, which would provide the host with resistance to a specific antibiotic if the uptake is successful. Then, by plating the cells on the same antibiotic, cells that are able to grow would indicate successful transformation.


Inserted Gene

This will be a foreign gene inserted into the plasmid for genetically engineering the bacteria.


Constructing a Plasmid


QGEM – 2019 Project Goal

QGEM 2019 hopes to express recombinant antibodies in E.coli bacteria using engineered plasmids. Antibodies do not naturally exist in bacteria and therefore we are only expressing specific fragments to avoid troubles in protein folding.
We are expressing Fab fragments and short chain variable fragments:


Figure 4: Antibody Fragments [5]

The diagram above shows different types of antibody fragments [5]. Light blue and dark blue labels the light chain and heavy chain respectively.


Our Plasmid: pET-duet

There are many types and versions of plasmids available, and researchers often have to pick one that suits the nature of the project. The plasmid we are using is called
pET-duet.

Figure 5. pET-duet Plasmid

For more information about this plasmid:

https://www.biocompare.com/Product-Reviews/40993-Co-expression-with-pETDuet-1-Duet-Expression-System-From-Novagen/

Plasmid Assembly: Gibson Assembly

One must introduce the target gene into the plasmid before introducing it to the bacteria. There are many methods, but QGEM is employing the Gibson Assembly technique. To assemble the DNA, the target gene will first be cleaved with an exonuclease on one strand at both ends to create 3’ overhangs. At the same time, the plasmid will be digested with restriction enzymes at specific sites. This way, the target gene and the plasmid overhangs will anneal to each other. Next, polymerases will incorporate nucleotides to fill in the gaps between the annealed DNA fragments, the fragments will be covalently joined together by ligases to create a recombinant plasmid.

Figure 6. Gibson Assembly. [6]

Transformation

Transformation refers to the process of introducing plasmids to a cell. To induce plasmid uptake in bacteria, one could either heat shock the bacteria culture and force slight opening in the bacterium membrane. Electroporation is another commonly used method that increases the permeability of the membrane by applying electric shocks to the cell culture. One could then plate the bacteria on a plate containing the antibiotic that the cells were given resistance to, and select colonies that have been successfully transfected.

References

[1] http://ulogs.org/@scienceangel/lab-diaries-7-creating-your-own-super-cool-strikekiller-mutant-strike-safe-to-use-virus-from-scratch-part-i. [Accessed: 05-Jun-2019].
[2] J. W. Sahl et al., “Insights into enterotoxigenic Escherichia coli diversity in Bangladesh utilizing genomic epidemiology,” Scientific Reports, vol. 7, no. 1, p. 3402, Jun. 2017.
[3] http://carlottachlaurenzi.blogspot.com/2018/08/charge-gateway-for-worldwide-customers.html. [Accessed: 05-Jun-2019].
[4] K. Morgan, “Plasmids 101: Origin of Replication.” [Online]. Available: https://blog.addgene.org/plasmid-101-origin-of-replication. [Accessed: 05-Jun-2019].
[5] https://www.biologicscorp.com/fab-fragment-antibody/. [Accessed: 05-Jun-2019].
[6] http://biomaster12.weebly.com/1/archives/02-2017. [Accessed: 05-Jun-2019].