In Vitro Synthesis And Fluorescence Spectrometry Biology Essay

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The notion of exploiting molecules which have been chemically labelled has revealed significant implication in chemistry, biochemistry and biology. The chemical labelling of bovine albumin with Rhodamine B isothiocyanate (RITC) allows for the production of fluorescent protein. Fluorophores (which is used in chemical labelling) covalently attaches to other molecules such as proteins, it contains a functional group which absorbs energy (exciting electrons) at the desired wavelength, causing it to fluoresce (due to excited electrons colliding which in turn returns the molecule to its ground state). The process of chemical labelling allows molecules such as proteins to be studied as well as having predominant role in immunohistochemistry.

The synthesis of green fluorescent protein (GFP) using RTS 100 E.coli Kit (through coupled transcription and translation) has overcome the problem with recombinant protein production which is toxic for organisms. In vitro transcription and translation GFP synthesis was performed using RTS 100 E-coli HY kit according to the manufactures instruction.

Introduction

Rhodamine B is an important congeneric form of Rhodamine dyes. It has the ability to fluoresce which can be measured using a spectrofluorometer. However, it has been found that Rhodamine B is a carcinogenic in vivo [1]. Rhodamine B isothiocyanate (RITC) (fig 1.0) is the fluorescent dye that was used for this experiment.

GFP which was first isolated from the jellyfish Aequorea Victoria [2] and consists of 238 amino acids which are arranged in beta barrel structure with fluorophore situated in the centre of the structure [3]. This conformation allows generation of visible fluorophores without co-factors. The use of GFP's has allowed radical advances in fluorescence microscopy, bioluminescence and extends to transgenic art.

The aim of the first part of the experiment was to attempt to produce a fluorescent protein by chemical labelling of albumin (extracted from chicken egg white) with Rhodamine B isothiocyanate (RITC) using spin-column protein purification. The second part of the experiment was to produce green fluorescent protein by coupled

transcription and translation system based on E coli lysate using a RTS 100 E.coli HY kit.

The labelling of biological molecules with fluorescent reagents has allowed extensive research into tracking and analysing the targeted molecules and chemical pathways [4]. Moreover, Fluorescent applications also extend to DNA and protein microarrays, photobleaching and the practice of using Fluorescence resonance energy transfer (FRET) to detect protein- protein interactions in vivo and vitro [5]. Using a Perkin Elmer LS50B spectrofluorimeter, measurements of the excitation and emission of the fluorescent protein allowed the graphical representations to be produced showing the excitation and emission max.

The chosen method allowed us to gain an insight into spectrofluorimeter. In addition to gaining experience with how to chemically labelled albumin with fluorescent dye and synthesis GFP in vitro. Moreover, spectrofluorimetry allows for the analysis of fluorescent molecules such as GFP in an efficient way, whilst also being fairly simple to use. Moreover, spectrofluorimetry allows detection over a wide range of wavelengths.

Materials and Method

Materials:

Bovine albumin (10mg/ml) solution. Rhodamine B isothiocyanate (10mg/ml). 10mM sodium bicarbonate buffer (pH- 8.5). Micro Bio-Spin 6 columns. RTS 100 E.coli HY kit in vitro purchased from Roche. Thermal block. Quartz cell. A Perkin Elmer LS50B spectrofluorimeter was used to produce excitation and emission spectra. Reference should be made to second year BS2570 Practical book (pages 3-14) for further information on the materials used.

Method:

the stock solution (albumin) was diluted. 4µl of albumin was added to 40µl of water to make solution A. 4 µl was extracted from solution A and added to 40 µl of water, producing the diluted sample of albumin. Albumin (10mg/ml), 10mM sodium bicarbonate buffer (pH- 8.5) and Rhodamine B isothiocyanate (10mg/ml) where added to four microcentrifuge reaction tubes labelled with unique group ID using a Gilson-200 automatic pipette to make a total volume of 40 µl in the manner shown below.

Reaction

Albumin (µl)

Buffer (µl)

RITC (µl)

Total volume (µl)

LBA1

2

34

4

40

LBA2

-

36

4

40

LBA3

2

38

-

40

LBA4

-

40

-

40

The reaction tubes where then placed on a thermal block and allowed to incubate for 1 hour at 25ËšC. The reaction tubes (LBA1 and LBA2) where then removed from the thermal block and purified using micro bio-spin chromatography columns and the

new reaction tubes where labelled LBA5 and LBA6, references where made to the practical book page 15-16 for instructions on how to use spin columns. After the

purification process, the reaction tubes where transferred to a fridge set at 4ËšC for collection the next day.

The second part of the experiment was to produce green fluorescent protein. Microcentrifuge test tube was taken and labelled with a unique group ID (SRI) and in vitro translation & translation mixture was prepared using a Gilson-20 micro pipette at room temperature to make a 25 µl solution. References should be made to the table in the practical book page 6. Moreover, the instruction manual for RTS 100 E.coli HY kit in the practical book should also be reviewed. The reaction tube was then placed into a thermal block and allowed to incubate for 12 hours at 30˚C.

The procedure for using the Perkin Elmer LS50B spectrofluorimeter allowed the excitation and emission spectra of fluorescent proteins to be measured. 1ml of 10mM sodium bicarbonate was added to the reaction tube LBA5 using a Gilson-1000 automatic pipette. The solution in reaction tube LBA5 was transferred into a quartz cell and placed in the spectrofluorimeter. The protocol was the followed according to the practical book page 7-8 and graphical representation of excitation and emission spectra where produced and saved onto a word document.

The GFP which was synthesised using coupled transcription& translation from day 1 was prepared for spectrofluorimetery. Using a Gilson-1000 automatic pipette, 1 ml of 10mM sodium bicarbonate was added to a 1.5ml tube. All the GFP was then added to the 1.5ml tube. The protocol was followed according to the practical book page 10-11 and the excitation and emission spectra of GFP was produced and emission and excitation max determined.

Results

Graph 1.0 - Green line shows the emission max spectrum which was 590nm for fluorescently labelled albumin. Blue and red lines are artefacts.

Graph 2.0 - blue line shows the excitation spectra and the excitation max which was 560nm for fluorescently labelled albumin.

Graph 3.0 - blue line show the emission max for population 1 which was 520nm, pink line shows the emission max for population 2 which was 590nm.

Graph 4.0 - blue line corresponds to the excitation max for population 1 of GFP which was at 370nm. The red line indicated that the wavelength for population 2 of GFP was 450nm.

Graph 5.0 - Using the excitation max for population 1 and 2 of GFP, the emission max spectra was taken again to prove that GFP was in two different forms. Purple line shows the emission max for population 1 which was 420nm (using the excitation max at 370nm). Green line shows that the emission max for population 2 was 590nm (using excitation max at 450nm)

Summary of results

Sample

Emission max (nm)

Excitation max (nm)

Emission max using excitation max (nm)

Albumin + RITC

590

560

-

GFP population 1

520

370

420

GFP population 2

590

450

590

Table 2.0 - shows a summary of emission and excitation max spectra for albumin and GFP.

Discussion

Background information:

Green fluorescent protein (GFP) which is derived from Aequorea Victoria is used in fluorescent detection of proteins. The ability for GFP to fluoresce without co-factors or incubating with a fluorescent dye [6] allows it to be more efficient than other techniques such as fluorescent microscopy.

Albumin is a protein which circulates in blood and mammals synthesis it in the liver. Bovine serum albumin (BSA) which is a form of serum albumin has three fluorphores (tryptophan, tyrosine and phenylalanine).

Fluorescence is a property that certain molecules posses and allows them to absorb light of a certain wavelength and to emit is as light and heat. A spectrofluorometer (fig 2.0) is an instrument which allows for the excitation and emission wavelengths of molecules to be measured.

Analysis of results:

Graph 1.0 show that the emission maximum for the chemically labelled albumin with RITC was 590nm. Comparing this result with the reference result for RITC which is 595nm [7], the results we obtained are very close to the reference value which shows that RITC successfully bonded to albumin. However, from graph 1.0, it is clear to see that at maximum emission the intensity is lower than it should be. The cause of this deviation was due to the small quantity of albumin+RITC which was caused by pipetting errors during the transfer of solution. The temperature is also a factor which could have contributed to the low intensity. As the temperature increases the intensity also increases. There is a positive correlation between the two factors.

Graph 1.0 also shows two artefacts, the possible suggestion for this is that there was an interference and the wrong wavelengths where chosen resulting in the samples emitting too much light.

By using the emission maximum as a fixed wavelength, graph 2.0 shows that the excitation maximum from the labelled albumin was recorded at 560nm. The reference wavelength for Rhodamine B was 547nm [8]. Similarly to the emission spectrum, the wavelength that was noted during the experiment was very close to the reference wavelength of RITC which suggests that the results are accurate.

Due to the limited time we had, we were unable to carry out the measurement for reaction tube LBA6. This reaction had to be carried out to show that RITC successfully attached to albumin.

Graph 3.0 shows the emission spectra for GFP. GFP was observed to have two populations, population 1 had an emission maximum 520nm and population 2 had an emission maximum at 590nm. There are several factors which might explain why there where two populations in the solution. Firstly, this could have been because the some of the GFP was folded and some unfolded which would have given two different structures and would have therefore emit light at different wavelengths. Another explanation could have been because the solution contained Wild type GFP. This is because the spectra for WTGFP closely resembled that of our GFP [9]. This is because in wtGFP, there are two different types of fluorophores which get excited at different wavelengths.

Graph 4.0 was produced by inputting the fixed emission max for population 1 and population 2 to obtain the excitation max. Graph 4.0 further confirms the existence of 2 types of GFT.

Graph 5.0 - Using the excitation max for population 1 and 2 of GFP, the emission max spectra was taken again to prove that GFP was in two different forms. A further explanation for the 2 different types of GFP could be because of the absence of a regulatory system, because there is no regulation in how the protein folds, there could have been some errors resulting in the formation of different structural arrangement of GFP and which resulted in two different emission max peaks.

The method adopted for this experiment to create GFP using RTS 100 E.coli HY kit was an improved method to recombinant protein production.

Conclusion

The method of producing fluorescent proteins whether it is by using a fluorescent die or synthesising it in vitro allows for the tracking and structure conformation to be analysed. This experiment allowed for student to gain experience producing fluorescent biopolymers and using spectrofluorimeter to study the emission and excitation spectra. The results obtained where accurate and matching the reference values. Moreover, two populations of GFP where found which had different emission and excitation spectra.

Additional questions:

Why controls have to be used in the study of labelled albumin?

Controls are a vital aspect to all experiment and all experiments should have a control. The reaction tube which contained only buffer and RITC was the control for albumin and it was used to observe how RITC combined with albumin, and to show that albumin was required for it to fluoresce. The reaction tube which contained only albumin and buffer was the control for RITC, which was used to show that without RITC albumin would not fluoresce.

Which controls would you add for the GFP experiment?

The experiment for GFP should also be repeated without GFP (control plasmid DNA), as this would show if the coding for GFP is what code for the protein GFP and subsequently allowing it to fluoresce.

Which other labelling/ detection techniques would you use with biological molecules if fluorescent labelling was not available?

One method which allows for biological molecules such as proteins to be detected/ labelled is by in vivo synthesis. This method requires E.coli to grow in tD2O, 15N- ammonium salts and 13C-glucose. The proteins that the E.coli produces will be labelled with 15N and 13C. However, one limitation to this method is that as protein sizes increase, it becomes more difficult to interpret the NMR spectra. Moreover, the process of labelling proteins with this method is directly affected by the ability for the uptake of isotopes. One advantage to this method is that is allows proteins of less than 40kD to be analysed and the three dimensional structural arrangement to be solved.

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