Can the E. coli bacteria expresses the beta-galactosidase enzyme if grown in a media containing lactose versus glucose?

Introduction 

The main purpose of this research project is to investigate whether the E. coli bacteria can express the beta-galactosidase enzyme if grown in a media containing lactose, MacConkey agar, versus a media containing glucose and trypticase-soy agar. In the E. coli bacteria, lacZ gene is one of the structural genes responsible for β-galactosidase (Smith et. al. 2005) . β-galactosidase is essential in the cells of an organism since it forms the fundamental provider in the process of generating energy as a major source of carbon from the process of lactose breakdown to form galactose and glucose (Pääbo, 2010). The E. coli on the other hand is essential in molecular biology practical because of its ability to divide faster and its genetic components to multiply faster. E. coli bacteria is capable of causing a regulated gene expression essential for the metabolism of lactose. The E. coli bacteria grows faster when placed in a glucose culture while in a lactose culture, the bacteria grow at a slower rate. The experiment aims at to determine if the E. coli bacteria expresses the beta-galactosidase enzyme if grown in a media containing lactose, MacConkey agar versus a media containing glucose and trypticase-soy agar (Porebski et. al 2014). 

Materials used 

20 mM Tris·Cl, pH 8.0 (pH buffer) 

2 mM sodium EDTA (chelating agent) 

1.2% TritonX-100 (detergent) 

Lysozyme 20 mg/ml final concentration (enzyme to break bacterial cell walls) 

Procedure 

DNA Extraction 

Add 1000 μl of bacterial sample into separate Eppendorf. 

Now centrifuge these at max speed for 2 minutes or until pellet forms. Discard supernatant (the liquid portion), without disturbing the pellet. 

Add 180 μl Enzymatic Lysis Buffer to the pellet and resuspend by pipetting up and down several times. 

Incubate for 30 min at 37°C in the water bath. 

Add 25 μl proteinase K . Proteinase K digests proteins. 

Add 200 μl Buffer AL without ethanol (ensure that ethanol has not been added to Buffer AL). Buffer AL contains guanidine HCl, a chaotrophic salt. It is also an irritant to bare skin so wear gloves. 

Incubate in 56°C water bath for 30 min. 

Add 200 μl Ethanol (100%) to the sample, and mix thoroughly by vortexing. 

Pipette the mixture from step 7 into the spin column placed in its 2 ml collection tube. Centrifuge at 6,000 x g for 1 min. Discard flow-through and collection tube . KEEP the silica column part which contains DNA bound to it. 

Place the silica column in a new 2 ml collection tube, add 500 μl Wash Buffer 1 , and centrifuge for 1 min at 12,000 x g. Pour out flow-through into waste beaker and replace silica column in collection tube. 

Add 500 μl Wash Buffer 2 , and centrifuge for 1 min at 12,000 x g. Pour out the flow-through and replace the column in the collection tube. 

Centrifuge the column with no liquid for 2 min at 12,000 x g to dry the silica matrix. 

Following the centrifugation step, remove the spin column carefully so that the column does not come into contact with any remaining flow-through. 

Place the silica column in a clean 1.5 ml microcentrifuge tube. 

Pipet 200 μl Elution Buffer (TE or Tris-EDTA buffer) directly onto the silica matrix. Hold at room temperature for 1 min, and then centrifuge for 1 min at 12,000 x g to elute. 

Remove silica column and discard it. Cap the 1.5ml microcentrifuge tube containing the eluted DNA. Store the amplified DNA at -20 o C in the LABELED 1.5 ml microcentrifuge tube. 

Polymerase Chain Reaction (PCR) 

You are going to prepare a master mix for 4 reactions per two-person groups . 

Once master mix is ready, aliquot 45ul each into ‘3’ PCR tubes and label them. The 4 th reaction was a slop and you don’t have to aliquot that. 

The 3 reactions would be (+ control partner #1’s DNA, + control partner #2’s DNA, - control mutual for both partners). 

Don’t forget to add 5ul of DNA into your positive control PCR tube. This is the same DNA that was isolated from the bacterial colonies in step I – DNA extraction. 

Place the PCR samples in the thermocycler and wait for further instructions. 

Protein Extraction and SDS-PAGE 

10 ml of Cell culture was centrifuged for you at 7000 rcf for 5 minutes at room temperature done for both Lactose and Glucose grown samples. 

Decant the supernatants carefully by pipetting, be sure not to disturb the pellet. Resuspend the cell pellet in 1 ml of Lysis buffer (on ice) by pipetting up and down. 

Transfer this mixture to a clean 5 ml Falcon snap cap. 

Sonicate each of the samples (must be on ice), for a total of 3 times, each time sonicating for a pulse of 10 seconds, then waiting 45-60 seconds before the next pulse to prevent overheating. Lysis is complete when the cloudy suspension becomes translucent, avoid frothing! 

Transfer samples to clean and labelled 1.5 ml eppendorfs. 

Centrifuge for 5 minutes and maximum speed (this separates the soluble proteins in supernatant from the insoluble pellet proteins) 

Transfer the supernatant to a new clean labelled 1.5 ml Eppendorf and discard the pellet. 

Transfer 15 uL of the sample to a PCR tube. 

Add 5 uL of 4x concentration Loading Buffer to the PCR tube, close it and mix by finger flicking. 

Heat at 99C for 5 minutes in the thermocycler. 

Load all the sample in the PCR tube onto SDS-PAGE gel. 

Don’t forget to leave one well in the SDS-PAGE gel for a protein ladder to compare your results with! 

Run the SDS-PAGE gel at 100-150 volts for about 45 minutes. 

Disassemble gel and stain in Coomassie blue for 30 minutes on a shaking incubator at approximately 150-200 speed (gel is usually placed in a staining tray). 

Discard the Coomassie blue carefully without damaging the gel. 

Add Destaining solution to the tray until the gel is submerged. 

Leave the gel on the shaking incubator overnight and the gel would be ready for visualization! 

RNA extraction and purification 

Cells: harvest cells by taking 1 ml of each Lactose and Glucose cells into 2 separate eppendorfs and centrifuge them at max speed for 1 minute. Discard the supernatant without disturbing the pellet. 

Lysis: to the pellet, add 600 uL of Buffer RLT and mix by pipetting up and down, and wait 1 minute. Centrifuge for 3 minutes at maximum speed. DON’T throw the supernatant as you will need it for step 3. Transfer it into a clean new Eppendorf. 

Add 600 uL of Ethanol to the supernatant from step 2 and mix well by pipetting and immediately move to step 4. 

Transfer up to 700 uL of the mix from the previous step to an RNeasy mini spin column (placed in a collection tube), Close the lid and centrifuge for approx. 15 seconds at 8000g. Discard the flow through. 

Add 700 uL Buffer RW1 to the spin column. Close lid and centrifuge for 15 seconds at 8000g. Discard flow through. 

Add 500 uL Buffer RPE to the column. Close lid and centrifuge for 2 minutes at 8000g. 

Drying: Replace the ‘collection tube’ with a new one and dry spin for 1 minute at 8000g. 

Elution: Place the RNeasy spin column in a clean Eppendorf and add 40 uL of sterile water (h 2 o). Close the lid and centrifuge for 1 minute at 8000g to elute the RNA. 

Save the RNA in -80 freezer for next use. 

Results 

DNA extraction 

PCR amplification of beta-galactosidase gene followed by gel electrophoresis 

RNA extraction and RT-PCR 

Protein extraction followed by gel electrophoresis and coomassie blue staining 

Discussion 

Lac operon in the E. coli bacterium for a set of four essential genes that work together in allowing the E. coli bacterium to utilize lactose as a source of energy. The Operant forms a classification of genes that are co-transcribed using one single mRNA that is regulated from the promoter. Operons are mostly found in bacteria such as the E. coli but at times exists in viruses (Pitcher et. al 2008). There are two essential genes that the cell uses to synthesize lactose. The beta-galactosidase, an enzyme that is coded by the lacZ, is responsible for the breakdown of lactose to yield D-galactose as well as the D-glucose. LacL is one of the repressor gene which codes and hinders the lac operon form undergoing transcription through the RNA (Cenis, 2009). The repressor gene bids to the allactose which is a derivative of the lactose material and causes itself to be transcribed by the chromosomal DNA thus giving a chance for the lac gene to be expresses. The research project can be improved further in the future through a collaborative research among scientists and intense molecular experiments. 

References 

Cenis, J. L. (2009). Rapid extraction of fungal DNA for PCR amplification. Nucleic acids research , 20 (9), 2380. 

Pitcher, D. G., Saunders, N. A., & Owen, R. J. (2008). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Letters in applied microbiology , 8 (4), 151-156. 

Porebski, S., Bailey, L. G., & Baum, B. R. (2014). Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant molecular biology reporter , 15 (1), 8-15. 

Pääbo, S. (2010). Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification. Proceedings of the National Academy of Sciences , 86 (6), 1939-1943. 

Smith, A. C., Stewart, R., Shields, P., Hayes-Klosteridis, J., Robinson, P., & Yuan, R. (2005). Introductory biology courses: a framework to support active learning in large enrollment introductory science courses. Cell Biology Education , 4 (2), 143-156.