Ultraviolet light inactivation of Escherichia coli cells expressing sugar beet (Beta vulgaris ) plant hemoglobin (BvHb1.2)

Abstract

Hemoglobin (Hb) is a protein, composed of a heme prosthetic group with an iron atom in the center of the protoporphyrin ring and the iron is the main component that bind oxygen. Hb was first found in the red blood cells of vertebrates and after in prokaryotes, fungi, plants and animals. In plants, there are three main groups of Hb proteins which are symbiotic, non-symbiotic and truncated. Non-symbiotic Hbs (nsHbs) are divided into two classes, class I (nsHb1) and class II (nsHb2). The aim of this study was to inactivate the E.coli cells expressing BvHb1.2 protein which is a nsHb1 from sugar beet (Beta vulgaris) by using the UV light. The recombinant protein was expressed in E.coli BL21 (DE3) and the cells were produced. The SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) and western blot were done to test if the BvHb 1.2 mutants were really expressed. The UV-C treatment of E.coli cells containing BvHb 1.2 was evaluated at various optical density measured at 600 nm (OD600) ranging from 1 to 7.5 within two hours of irradiation on a small (572.265 cm2) and big petri-dish (1256 cm2).The UV-C irradiation of red blood cells (RBCs) as well as the E.coli expressing human Hb was also done for 10 min to evaluate the effect of UV-C on Hbs. Thereafter, the irradiated cultures were plated on LB medium with and without ampicillin and bacteria were enumerated after 18 hours of incubation. The extracted BvHb 1.2 protein, Human Hb and Hb from RBC before and after UV irradiation were conveniently analyzed by using the carbon monoxide (CO) assay. Since, the carbonmonoxyHb complex of BvHb 1.2 shows two absorption bands with their maxima near 537 and 566 nm and also at 416 nm while carbonmonoxyHb complex of human Hb shows their absorption band at 419 nm, 539 nm and 569 nm. The oxy form and deoxy form of Hb were also examined. This study showed that there was a linear correlation of the optical density and the inactivation time and the effective bactericidal effect of UV-C was found at OD600 ranging from 1.3 to 1.95 for 10 min of irradiation on a big petri-dish. There was no significant difference in plating the irradiated cultures either on LB medium without ampicillin or with ampicillin (100 mg/ml) and there was also no remarkable UV effect on the state of Hb for BvHb 1.2. However, E.coli cells expressing human Hb was destroyed after UV irradiation.

Popular Abstract

Hemoglobin (Hb) is an essential component of the red blood cells (RBCs) mainly found in vertebrates. The main function of the Hb is to transport the oxygen from lungs to tissues. Moreover, Hbs also bind other gaseous ligand like carbon monoxide, nitric oxide and organic molecules which makes them multifunctional proteins in living organisms. In plants, Hbs were divided into three broad groups after comparing their evolutionary relationship and oxygen-binding properties. These Hb groups are symbiotic Hb (sHb), non-symbiotic Hb (nsHbs), and truncated Hb (trHb). The sHbs (also known as leghemoglobin) undergo symbiotic association with microorganisms where the plants benefit from the nitrogen-fixation carried out by their bacterial partners, and in turn help to provide oxygen to the respiring symbiotic bacterial cells in a manner analogous to hemoglobin transporting oxygen to respiring tissues in animals. On the other hand, nsHbs are found in all plants and are divided into two classes; class I and II. Class I nsHb have an extremely high affinity for oxygen while Class II nsHb are more closely related to the sHb and they have lower affinity for oxygen. However, the function of nsHbs in plant tissues has not been established. The first study of the expression of nsHbs in sugar beets revealed three different nsHb genes. They are BvHb 1.1, BvHb 1.2 and BvHb 2 and the analysis of their protein sequences showed that both BvHb 1.1 and BvHb 1.2 nsHb genes belong to Class 1 and BvHb 2 to Class 2.

Hb can be used as a blood substitute due to its oxygen carrying capacity and one way of increasing the production of Hb is to express it in E.coli. Today, million tons of sugar beet are produced in Europe and Sweden and if sugar beet Hb is extracted and also used as a blood substitute that could save thousands of human lives worldwide. The bacterial effect of UV light is known for more than a century and many studies have shown that it is possible to kill different E.coli strains by using short wave ultraviolet (UV-C). In this thesis, the UV inactivation of E.coli cells expressing BvHb1.2 protein which is a nsHb1 from sugar beet (Beta vulgaris) was studied on a small (572.265 cm2 ) and big petri-dish (1256 cm2). The UV inactivation of red blood cells (RBCs) and human Hb expressed in E.coli cells were also evaluated. Thereafter, the irradiated cultures were grown on plates containing LB medium with or without ampicillin and bacteria were enumerated after 18 hours of incubation. Their capacity to bind carbon monoxide was analyzed before and after UV irradiation. It was found that BvHb 1.2 was not affected by the UV irradiation while E.coli cells expressing human Hb was destroyed after UV irradiation.

• author: Ishimwe, Florence
• supervisor: Nélida Leiva
• organization: Pure and Applied Biochemistry
• course: KBKM01 20191
• year: 2019
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: Ultraviolet light, Escherichia coli, sugar beet(Beta vulgaris), non-symbiotic plant hemoglobin, BvHb 1.2., Applied biochemistry, Tillämpad biokemi
• language: English
• id: 8983761
• date added to LUP: 2019-07-04 14:29:19
• date last changed: 2019-07-04 14:29:19