What Are Antibiotics?
The definition of an antibiotic is an agent that kills bacteria or inhibits their growth. There was once a time when antibiotics were produced from natural sources. However, today, the most commonly used antibiotics are either semi-synthetic or fully-synthetic compounds. Antibiotics can be divided into two categories, bactericidal, or bacteriostatic. When an antibiotic is bactericidal it kills bacteria directly, when an antibiotic is bacteriostatic it reduces growth or prevents cell division. It can be difficult to differentiate antibiotics using these categories as when they are in high concentration bacteriostatic antibiotics can kill bacteria and bactericidal antibiotics can slow down the growth of bacteria when used in high concentrations.
Sometimes researchers can take for granted how much easier the use of antibiotics makes their work. Plasmid transformation into E.coli is not the most efficient procedure, and if there wasn’t a way of finding out which cells received the right plasmid then it would take scientists significantly longer to find the right clones for the cells. Bacterium is at a disadvantage when there is a presence of plasmid, a plasmid-containing cell needs to replicate the plasmid but it also needs to replicate its chromosomal DNA, resulting in additional resources being necessary to keep the plasmid intact. By adding an antibiotic resistance gene to the plasmid you kill two birds with one stone. Scientists can detect plasmid-containing bacteria when the cells are grown on selective media, and the bacteria are provided with the pressure to maintain their plasmid.
Other Uses of Antibiotics in the Lab
The disruption of genes at the chromosomal level is another application of antibiotics in the lab. Scientists will try to interrupt or destroy a gene by trying to allow an antibiotic to enter the coding region of the gene. This disrupts the gene and serves as an indication of mutation. The loss of resistance also indicates that cloning has taken place successfully. When this occurs, there are two separate resistance cassettes that the cloning vector will usually have, your gene of interest will either completely clone into or remove one cassette. The removal of one cassette is categorised as Gateway cloning. In the case of counterselection, a researcher will only select the bacteria of the antibiotic that is maintained.
There are a few factors that you can take into consideration when choosing research antibiotics. The cells you wish to maintain and which antibiotics will be effective on the cells you don’t wish to maintain are the key factors that should help you choose the most appropriate antibiotic for your research.
Ampicillin
So where does Ampicillin come in? Ampicillin is a semi-synthetic antibiotic of the beta-lactam class. Beta-lactam class antibiotics are broad spectrum, and they can be identified by their molecular structures. Included in the beta-lactam class are every single penicillin derivative, cephalosporins, carbapenems, and monobactams. By joining the beta-lactam ring to the penicillin-binding proteins the majority of beta-lactam antibiotics can hinder cell wall synthesis. This results in not only instability of the cell wall but also the cell wall becoming lyses. Beta-lactamases are enzymes created by bacteria that cause the disintegration of the functioning beta-lactam ring and allow for the resistance of this class of antibiotics.
Out of all the broad-spectrum beta-lactams, ampicillin is one of the most regularly used. It is made up of a thiazolidine ring and a side chain that is linked to the beta-lactam ring. In the selection experiment, ampicillin is used to help select resistant bacteria. Also, when it comes to prokaryotic selection experiments, ampicillin is extremely useful.
How to Solve the Limitations Associated with Ampicillin
While resistance to ampicillin can be a useful tool as a selection marker for plasmids in gene cloning and protein expression in E. coli and other bacteria, it does have some disadvantages you need to take note of if you are planning to use it. For example, it is important to make use of ampicillin plates within four weeks to optimise their utility, as they are highly perishable.
Beta-lactamase expressed through the plasmid-borne bla gene that causes the hydrolosis and inactivation of ampicillin is the basis of ampicillin selection. The problem with beta-lactamase is it is also emitted by bacteria into the medium. If the culture is not managed correctly, the ampicillin in the culture medium can be made inactive and remove the selective pressure. This would come as a result of the build-up of extracellular beta-lactamase.
If this occurs, plasmid might not be present in a large portion of the cell, and protein expressions and poor plasma preps might suffer as a result of this. On agar plates, satellite colonies can be formed when ampicillin deteriorates. Satellite colonies are cells that are very small and have not taken up the plasmid that appear around a large colony that has taken up the bla- which has the plasmid inside. The beta-lactamase that is given off by the colony that is expressing the bla- deteriorating the ampicillin which is in close proximity of the colony is what causes the formation of small colonies.
When moved over to a medium that contains ampicillin, the satellites will grow, meaning they will not cause any issues.
How to Avoid Plasma Loss
Beta-lactamase build-up might be the cause of poor yields in your plasmid preps and unsatisfying protein expression. However, when using ampicillin as a selection marker there is a way to prevent plasmid loss from happening:
- Make sure not to let liquid cultures saturate for too long.
- Take away beta-lactamase that is released from starter cultures. Before inoculating the main culture, pelleting and re-suspending the starter culture in a fresh medium that is absent of an antibiotic.
- Use a higher concentration of ampicillin if you are running into issues. This helps with avoiding satellite formation and makes it more difficult for beta-lactamase to inactivate all the ampicillin.
- To avoid using less effective ampicillin concentration, don’t use outdated ampicillin.
While ampicillin has its disadvantages, it can be highly effective and there are ways of managing its limitations. If you are looking for products to solve your antibiotic needs in the lab you can look through our range of supplies. You also have the option of contacting one of the members of our team who could guide you in selecting the right products for your lab work.
