Difference between revisions of "Part:BBa K1927002"

Latest revision as of 18:35, 21 October 2021

β � lactamase AmpR

This gene conveys resistance to a common antibiotic called Ampicillin. In research it is often used as a selection marker

Usage and Biology

During the last decade antibiotic resistance has grown not only in incidents but also in awareness among the public. Rapid detection of these incidents is essential in the battle against sophisticated bacteria. Beta lactamases are enzymes that provides one of the many mechanisms of antibiotic resistance. Its for this reason regulary used among researchers as a selection marker. Bacteria that has been subjected to a procedure where foreign DNA is introduced, an antibiotic resistance gene is often use to check if colonies succsesfully has taken up the DNA and expressed it.

An example of this is ampicillin which is a highly used selection marker. The need for a selection marker in genetical engineering has been shown extremly usefull. By transforming a bacteria with a particular gene plus an antibiotic resistance gene it allows the researcher to know exactly which colonies that has taken up the DNA. By plating them out on agar plates with a given antibiotic, only the bacteria with the resistance gene can grow. Cells that did not manage to take up the DNA will die.

The mechanism of beta lactamases are oriented to the bacterial cell wall. This cell wall is unique to bacteria and consist of several components. Gram Positive and gram negative bacteria will have a different cell wall composition. In general, Gram-positive bacteria have a thicker layer of cell wall as well as a layer of cytoplasmic membrane. These layers consist of several conserved compounds such as monomeric disaccharide tetrapeptide, which are usually also those that will trigger an immunological defence respons of the host. Gram-negative bacteria (e.g., Escherichia coli) typically contain an outer membrane, an intervening periplasmic space where a thin layer of cell wall resides, and a layer of cytoplasmic membrane. Beta lactamases are usually produced both by gram negative and positive bacteria, either from plasmid or chromosomally. Beta lactamases are able to resist several types of antibiotics. These antibiotics all have in common a 4 - atom ring called beta lactam ring which the enzyme are able to hydrolyze and break open and the molecule looses its antibacterial function.

Penicillin, a regulary used antibiotic have such a beta lactam ring. This drug was the first antibiotic to be discovered and is still widely used today. This ring will bind to an enzyme (DD –transpeptidase) that is in charge of renewing the bacterial cell wall. Without this enzyme there will be no new formations of peptidoglycans for the cell wall and the integrity of the bacterial cell wall will be lost, it will eventually rupture and the bacteria will die. By hydrolyzing the ring, it will make the molecule unable to bind to the cell wall producing enzyme, thus the Penicillin have lost its destructive activity.

Characterization of BBa_K1927002

Group: TJUSLS_China 2019
Author: Dongxu Li
Summary: AmpR，a class A beta-lactamase, is the most widely used selective marker in gene manipulation. It can hydrolyze ampicillin with specificity. Our team, TJUSLS_China, intends to screen inhibitors of the class B metallo-beta-lactamase. Details visible to our wiki:[1]. Due to our project requirement, we want to prove the difference hydrolysis capacity on antibiotic substrate of class A beta-lactamase(take AmpR as an example) and class B metallo-beta-lactamase(take NDM-23 as an example). So we did the contrast experiment of the two enzymes. We chose faropenem, meropenem and cefazolin as the antibiotic substrate for the two enzyme. The data is as follows.

Figure 1. Hydrolysis of three different antibiotics of E. coli with AmpR gene(a) and E. coli with NDM-23 gene(b). The absorption values of faropenem, meropenem and cefazolin at different nanometer wavelengths were measured at 307nm, 300nm and 273nm, respectively. The concentration of every antibiotic was 250 μM. The OD600 of E. coli with AmpR protein was 0.4, and the E. coli with NDM-23 protein was 0.02.

Figure 2. Hydrolysis of faropenem of E. coli with AmpR gene and E. coli with NDM-23 gene. The absorption values were measured at 307nm. The concentration of faropenem was 250 μM. The OD600 of E. coli with AmpR protein was 0.4, and the E. coli with NDM-23 protein was 0.02.

The results showed that the hydrolytic capacity of AmpR to all three antibiotics was lower than that of NDM-23, which proved that class B metallo-beta-lactamase had a broader substrate spectrum.

Generation of biobrick BBa_K1927002 and BBa_K1927003
We wanted to calibrate our detection test with a biobrick of our own, in addition to the positive control (E. coli amp), negative control (E. coli without antibiotic resistance) and the purified protein from biobrick BBa_K1189031 as an additional positive control.

We retrieved the gene sequence for the Amp-gene from the standard E. coli vector pUC19 . The AmpR gene product is a β-lactamase class A which confers resistance to ampicillin, carbenicillin, and related antibiotics. The gene was designed to include prefix, suffix and start of vector sequence to make it ideal for Gibson assembly later on. Gene and primers was synthetized by IDT. We performed a Gibson assembly with the shipping vector pSB1C3 and our gene to generate biobrick BBa_K1927002.

Figure 1: LB agar plate with chloramphenicol for growth of transformed bacteria(TOP10) with BBa_K1927002. The insert was inserted into the shipping vector pSB1C3 by Gibson assembly.

Figure 2: Gel analysis of colony PCR with BBa_K1927002. The insert was inserted into the shipping vector pSB1C3 by using Gibson assembly. Lane 10 are DNA ladder (GeneRuler 1 kb DNA ladder, Thermo Fisher Scientific). Lane 11 are positive control (validated part BBa_K1927000). Lane 18-19 are different colonies from Gibson assembly. They show the appropriate band at approx 1000bp.

This particular gene is collected from puc19 vector and is an ampicillin resistance gene. The

sequence was collected online and sent for synthesizing to IDT.

Several methods were discussed on how to assemble the biobrick, and the team chose to

try out Gibson Assembly. The method has not yet been tested out by the registry, but has

been tried out by several teams who were succsessfull.

Advantages with Gibson Assembly compared to the standard assembly with resitriction

enzymes and gel purification is several.

- First of all its fewer steps compared to classical cloning, with fewer steps will DNA

loss and also other risks be reduces as well as price. - It requires few components and few manipulations

- Its suitable for combining several fragments at once

- The whole process takes shorter time

- No restriction site scar remains between the two DNA fragments

Disadvantages

- Primers must be ordered ahead. - PCR may be tricky

The team used Snap gene to design the appropriate primers. Snap gene is a software that

allows you to visualize and design your DNA sequences, it is perfect for primer design as

you can visualize all of your sequences at once and place your DNA into your desired vector

by only pressing a few buttons. The primers were then ordered online at IDT.

To perform Gibson assembly, the amount of insert needs to be 2-3 fold in excess compared to amount of vector.

Usually the smaller the volume of the reaction the better.
Gibson assembly has

Contribution by iGEM Evry Paris-Saclay 2021

This part is AmpR, which is a derivative of the TEM-1 β-lactamase (Uniprot P62593) with two mutations V82I and V182A. Six sequences encoding it are found in the Parts Registry (BBa_K2082292, BBa_Y1002, BBa_I722014, BBa_K1927002, BBa_K2308011 and BBa_K2796014). BBa_K2082292 differs from all the other five by a synonymous mutation: T/C in position 132.

β-lactamases are enzymes secreted extracellularly by some bacteria. They allow the bacteria to resist β-lactam antibiotics by inactivating them before they can act (Figure 1). The best known β-lactamase to date is TEM-1, discovered in 1960, which allows resistance to penicillins and cephalosporins [1].

Figure 1. The mode of action of β-lactamases: hydrolysis of the β-lactam ring.

β-lactam antibiotics represent a large proportion of clinically used antibiotics. They are classified into different families based on their structure: penams (ex. penicillin), penems (ex. carbapenem), cephems (ex. cephalosporin) and monobactams (ex. aztreonam). These β-lactams have a bactericidal action [2]. Their mechanism of action involves crossing the peptidoglycan (PG) in the bacterial cell wall until they reach the enzymes called PBPs (penicillin binding proteins), which they inhibit thus preventing the synthesis of PG and promoting the release of an autolysin that will digest the PG. The mode of action of these β-lactams depends primarily on the presence of PBPs. Indeed, the efficacy of the antibiotic depends on the affinity it has to the PBP, the number of PBPs that the bacterium has on the surface of its peptidoglycan, but also their structure, which may differ, preventing the recognition of the PBP, as is the case with methicillin-resistant Staphylococcus aureus [3].

Ampicillin is a penam antibiotic that acts primarily on the cell-wall synthesis of Gram-positive bacteria, since the peptidoglycan is not protected by the outer membrane, and on some Gram-negative bacteria by using the process of passive diffusion through porins to reach the peptidoglycan. This broad spectrum antibiotic is widely used in molecular biology and has an application concentration of 100 µg/ml.

Carbenicillin, which belongs also to the penam family, is also used at a concentration of 100 µg/ml in molecular biology for selection in the laboratory.

The laboratory selection is based on the presence of a β-lactamase gene, the AmpR / bla, in the vector used to genetically modify bacteria, and it’s absence in the parental strain.

A panoply of vectors containing the AmpR gene are used to genetically modify bacteria. They have different origins of replications which define their copy number per cell and may influence the amount of β-lactamase produced and thus the amount of antibiotic a cell can resist.

We therefore were interested to know if the Gram-negative bacterium Escherichia coli could resist higher concentrations of Ampicillin or Carbenicillin, while taking into consideration the copy number of the plasmid harbouring the AmpR resistance gene.

To investigate this, E. coli NEB5ɑ cells were first grown overnight in 96-deep-well plates containing 1 mL of LB medium supplemented with 100 µg/mL ampicillin, then diluted by 40x into similar media. Upon reaching early log-phase, cells were further diluted 20x in 100 µL of LB medium supplemented with increasing concentrations of either ampicillin or carbenicillin (0 to 15000 µg/mL) in a transparent 96-well polystyrene microplate (Sarstedt). The plate was incubated at 37 °C at 200 rpm and ODsub>600nm</sub> was measured every 10 min for 24 hours in a CLARIOstar (BMGLabtech) plate reader. The ODsub>600nm</sub> values at the time point when the cultures performed in the absence of antibiotics reached the stationary phase were plotted as a function of the antibiotic concentration. These dose-response curves were fitted using the AAT Bioquest, Inc., Quest Graph™ IC50 Calculator to the sigmoidal équation:

where OD_600nm^min represents the minimal OD_600nm, OD_600nm^max represents the maximal OD_600nm, C is the concentration of the antibiotic, IC50 is the antibiotic concentration that inhibits 50% of the bacterial growth, Hill coefficient is describing the steepness of the curve. The OD_600nm values were converted into equivalent particles using the calibration curves presented on the "Measurement" page of our wiki.

The results obtained (Figure 2) for the median inhibitory concentration IC50 for the two antibiotics tested show us that E. coli bacteria harbouring the AmpR gene can resist 10 to 100 times higher antibiotic concentrations than those usually used in molecular biology techniques. When we focus on the inhibitory concentration as a function of the copy number provided by the plasmid (Figure 3), we observe no correlation, suggesting the resistance efficacy of the bacteria to ampicillin or carbenicillin is not closely dependent on the copy number of this gene per cell.

Figure 2. IC50 values of ampicillin and carbenicillin in E. coli NEB5ɑ cells harboring the AmpR resistance gene in plasmids with various origins of replication. The data and error bars are the mean and standard deviation of at least three measurements on independent biological replicates.

Figure 3. IC50 values of ampicillin and carbenicillin as a function of the AmpR gene copy number.

References

[1] Salverda MLM, De Visser JAGM, Barlow M. Natural evolution of TEM-1 β-lactamase: experimental reconstruction and clinical relevance. FEMS microbiology reviews (2010) 34: 1015–1036.

[2] Bush K, Bradford PA. β-Lactams and β-Lactamase Inhibitors: An Overview. Cold Spring Harbor Perspectives in Medicine (2016) 6: a025247.

[3] Chambers HF. Methicillin-resistant Staphylococcus aureus. Mechanisms of resistance and implications for treatment. Postgraduate Medicine (2001) 109: 43–50. Sequence and Features