Engineering the Human Growth Hormone into the TOPO TA Plasmid
Apply article — focus on gene editing and synthetic biology
By Izabela Ninu
About 0.02% of people in the USA have a Human Growth Hormone Deficiency (HGHD). The symptoms include short structure, poor growth, delayed puberty and mental development. It is especially relevant when talking about the development of children and teens.
This deficiency affects the quality of life of these children. Is there any way to produce this hormone synthetically?
1. The Human Growth Hormone
What is it?
Human Growth Hormone (HGH), also known as somatotropin, is a peptide hormone comprised of 191 amino acids (see picture 1). Synthesized and secreted by the somatotroph cells of the anterior pituitary gland, HGH holds a big significance in regulating growth, metabolism, and cellular regeneration.
Its primary function is to stimulate the synthesis of insulin-like growth factor-1 (IGF-1), which, in turn, mediates the growth-promoting effects of HGH. (see picture 2). During childhood and adolescence, HGH plays a central role in skeletal and organ growth, influencing the proliferation and differentiation of chondrocytes and osteoblasts. Moreover, HGH exhibits metabolic effects by enhancing lipolysis, mobilizing fatty acids, and promoting protein synthesis. While endogenous HGH production declines with age, the utilization of recombinant HGH has become a therapeutic intervention for growth disorders in children and is under investigation for various age-related conditions.
Recombinant Human Growth Hormone (rhGH) therapy has become a common treatment for growth-related disorders and various medical conditions. One prominent application is in addressing growth hormone deficiency (GHD) in children: pediatric patients with conditions such as Turner syndrome, chronic renal insufficiency, or Prader-Willi syndrome benefit from rhGH therapy to enhance growth and development, by administration of the synthetic or genetically engineered Growth Hormone.
Beyond pediatric applications, rhGH has been employed in adults with growth hormone deficiency to alleviate symptoms associated with this condition, such as decreased bone density and muscle mass, increased body fat, and reduced exercise capacity.
Athletes and bodybuilders have sometimes misused rhGH for its purported performance-enhancing effects, although this practice is controversial and potentially associated with adverse health effects.
In this article, we will focus on genetically integrating the Human Growth Hormone into an E. Coli plasmid, recreating the research on the Recombinant Human Growth Hormone.
2. How to transfer a gene into another organism?
A. Plasmids and their origin
A plasmid is a small, circular DNA molecule that is separate from the chromosomal DNA in the cells of bacteria and certain other microorganisms.
Unlike chromosomal DNA, which is essential for the organism’s survival, plasmids often carry accessory genes that provide additional functionalities, such as a foreign gene in our case.
Plasmids are capable of independent replication within the host cell, as such carrying a possibility to replicate the interested gene that we insert into it. This ability to replicate independently makes them valuable tools for the production of recombinant proteins, gene expression studies, and other genetic manipulations.
When we insert foreign genetic material into a plasmid, it becomes a vector: a vector is a general term for any carrier molecule that transports foreign genetic material into a host organism. Vectors can include plasmids, viruses, artificial chromosomes, or other DNA molecules used to transfer genes. In the case where we use a plasmid with a foreign gene in it, we are talking about a plasmid vector.
B. Plasmid vectors: how do they work?
The key parts of a vector plasmid include the origin of replication, selectable markers, multiple cloning sites (MCS), and regulatory elements.
The origin of replication dictates the plasmid’s ability to autonomously replicate within a host cell, ensuring the efficient propagation of the vector.
Selectable markers, often antibiotic resistance genes or green fluorescence genes, confer a survival advantage or a visible marker to host cells that have successfully incorporated the plasmid.
The multiple cloning sites serve as locations where foreign DNA can be inserted into the plasmid, allowing for the expression of specific genes.
Additionally, regulatory elements such as promoters and terminators control the transcription and translation of genes carried by the plasmid.
(see picture 4 )
These components collectively make vector plasmids indispensable tools in molecular biology, enabling the introduction, expression, and study of genes for various research and practical applications.
3. Designing a Vector
A. Types of restriction enzymes
Restriction sites are specific DNA sequences recognized and cleaved (cut) by restriction enzymes, a kind of “molecular scissors”. These enzymes create cohesive /sticky ends or blunt ends in the DNA fragments (see pictures 5 and 6 above).
Different restriction enzymes, such as EcoRI, BamHI, and HindIII, have specific recognition sites, and their compatibility ensures successful gene insertion into the plasmid. The cohesive ends created by restriction enzymes in both the plasmid and the gene fragment facilitate a seamless connection through the formation of hydrogen bonds (see picture 7). The choice of restriction sites is determined by factors such as the desired orientation of the inserted gene, the size of the DNA fragments, and the compatibility of the cohesive ends.
B. Online simulation of the vector and gene combination
In genetic engineering, the online simulation of combining plasmids and genes has emerged as a necessary asset, revolutionizing the way researchers plan and analyze their experiments. The National Center for Biotechnology Information (NCBI — https://www.ncbi.nlm.nih.gov/) database serves as storage of genetic data, housing diverse DNA sequences and annotations.
I will also be integrating into this process the Snap Gene: an online platform that facilitates the visualization, editing, and simulation of DNA constructs. (https://www.snapgene.com/).
The virtual simulation aspect is particularly advantageous, as it enables us to experiment with various combinations of plasmids and genes before embarking on the physical experiments in the laboratory.
4. Recreating an existing research
A. Sources
Finding relevant research that provides the plasmid and restriction enzymes that were used is extremely hard, as I tried to recreate online what was accomplished in a lab. The research 1 that I looked into proposing a protocol for integrating the HGH into an (unknown) plasmid, later to be introduced in bacterial cells and then into mice cells.
The second research, however, provided information regarding the integration of the hGH gene, isoform 1, into the TOPO TA vector: I replicated this research, with modifications to the restriction enzymes that were used.
B. Downloading gene sequences
The NCBI website has a various range of data sequences, and it is important to know the specific sequence that you are interested in: here, we are interested in the Homo Sapiens Growth hormone, the mRNA that will code for the exact hormone. It is important not to choose the HGH receptor or any other alternatives, as we are only interested in the sequence that will, once transcribed, produce the specific amino acid sequence. (We will download the mRNA code in FASTA format, so the text-based sequence.)
Furthermore, we are also interested in downloading the specific gene that encodes for our plasmid of interest: the TOPO TA plasmid. This time, we will use SnapGene’s website (https://www.snapgene.com/) to download the specific plasmid. I searched for the pCR II TOPO TA plasmid, and this was the version that appeared on the SnapGene website.
C. Using SnapGene’s Editor to import and visualize the sequences
First of all, I imported the two downloaded sequences into SnapGene: the plasmid and the DNA strand.
D. Choosing the right restriction sites
Now, you can see all of the terms that are included here along the lines: these are all restriction sites. Matching restriction sites on both the plasmid and the DNA fragment ensures a seamless fit, much like aligning puzzle pieces.
This compatibility is critical for the success of genetic manipulations, allowing us to precisely integrate desired genes into plasmids. The cohesive ends created by restriction enzymes facilitate the formation of hydrogen bonds, enabling a stable connection between the plasmid and the gene.
Here, I selected the common restriction enzymes that were close to one another on the map: the enzymes BstAPI and XcmI. The purpose is to find the enzymes that, in the plasmid, hold the least amount of nucleotides between them as possible.
E. Creating the vector
When creating the vector in SnapGene, you choose the restriction sites, and then you have a view of the resulting sequence at the end of the restriction site: in this case, we will have the following sequence at the XcmI restriction site:
…AAGGAT
…TTCCT
and the following sequence at the BstAPI site:
…GTC
…CAGCCC
We are in both cases in restriction sites that leave a sticky end: however, these ends are not compatible with the ends of our gene sequence of interest. As such, what we will do is remove the respective overhangs. Fortunately, the program allows us to do it directly by choosing the check case.
Finally, we insert the gene of interest in the plasmid that has, now, blunt ends:
This is how, in SnapGene, you create your vector that carries the gene of interest.
Here we can review the process, and see the actions we undertook to create our vector.
4. Conclusion
As such, the successful integration of the HGH gene into a plasmid not only showcases the precision of molecular biology but also hints at the transformative potential for therapeutic applications: by creating a plasmid capable of producing the human Growth Hormone, we can obtain this synthetic molecule by only genetic engineering of a vector. This can be incredibly interesting for medical administration and any other treatment that involves this hormone.
A. However… costs?
How much would such a plasmid cost to realize?
In the EUROFINS company of genetics (UK), a long strand of nucleotides would cost about 0.36 £/ base pair, so about 1,763 £ for our gene of interest, plus another 120 £ for the Plasmid that we are using.
As such, such a plasmid would cost about 1900 £ to realize, a price that is quite high to be scalable in the medical field — as such, a solution needs to be found! Will be working on that later…
