HONG KONG POLYTECHNIC UNIVERSITY B. SC. (HONS.

) APPLIED BIOLOGY WITH BIOTECHNOLOGY ABCT316 Experimental Approach in Molecular Biology and Biochemistry 20112012 Semester 2

GRP Promoter Activity Assay

Project Report
Tang Wai Man (10525360D)

Contents
1. 2. 3. 4. 5. 6. 7. Objectives Background Information Materials and Methodology Procedures Results Discussion Reference

Page
1 2 3 7 8 8 9

Tang Wai Man (10525360D)

2012 Sem 2 ABCT316 Project Report

1. Objectives
To insert GRP promoter sequence cut out from pGL3-GRP vector into pGL4 luciferase reporter vector, to transfect the recombinant plasmid into 293T/17 mammalian cells and thus to test the promoter activity with and without drug treatment by dual luciferase system.

2. Background Information
2.1 GRP GRPs (glucose-regulated proteins) in endoplasmic reticulum (ER) such as GRP78 and GRP94 can act as molecular chaperones in cells [1]. The expression of GRP78 will be activated under ER stress, like glucose starvation, to protect the cells from apoptosis [2]. GRP78 as an ER stress sensor is an important biological target of many different human diseases and treatments (e.g. cancer therapy) [2]. Also, the transcription efficiency of GRP genes and promoter activity are affected by strength of GRP promoter so study of GRP promoter activity can help to investigate the effect of drug treatment on disease related to ER stress. A part of the GRP promoter sequence located inside the polylinkers of the pGL3 vectors is shown in figure 2.1.

Figure 2.1 Nucleotide sequences of the -140 to -19 region of the human GRP78 promoter. Underlined part shows the TATA box [3].

2.2 Luciferase Reporter Vectors It consisting of firefly luciferase gene was used to analyze the factors affecting human gene expression. Vectors that had been used in this project included pGL3 and pGL4.
Table 2.2 Comparisons of pGL3 and pGL4.
Properties Generation Reporter gene expression Abnormal expression pGL3 Old Lower Higher pGL4 New Higher Lower Because it has been i) removed the regulatory elements, ii) reduced the number of transcription factor, and iii) done codon optimization for mammalian expression. Improved by destabilizing the luciferase genes Higher As it i) provides flexible options for luciferase genes and mammalian selectable markers, and ii) has polylinker and SfiI transfer scheme allowing and easy transfer from vector to vector

Temporal response Usability

Slower Lower

Figure 2.2 Genetic maps of pGL3 basic vector (upper) and pGL4 vector (lower). (Source: Promega, Technical Manual of pGL3 Luciferase Reporter Vectors; Technical Manual of pGL4 Luciferase Reporter Vectors)

2.3 E. coli as Competent Cell

OD600: Number of viable cells does not exceed 108 cells / mL ensure that E. Coli does not grow to a higher density

2.4 HEK 293T Cell It is the human embryonic kidney cell line which can express human GRP. It is usually used for transformation due to easy to handle for culture and transfection [4]. The T means the cell membrane consists of SV40 large antigen for easy transfection with plasmids of SV40 late poly(A) signal. 2.5 Transient Transfection Transient transfection assay should be used as it is rapid, simple, and easy to quantify perform promoter from the results compared with other assays, like stable transfection assay, transgenic assay and homologous recombination assay [5]. 2.6 Dual Luciferase Assay
It integrates the Firefly & Renilla Luciferase Assays [6]. It is a reporter gene system of high

sensitivity and relatively low cost comparing to other options such as systemises of chloramphenicol actyltransferase and -galactosidase. Luciferase genes are linked to GRP promoter so promoter activity is directly proportional to the luciferase activity. 293T cells produce luciferase as a reporter which cannot be found in human originally. After bioluminescent reactions of luciferase, luciferase activity is measured by luminometer. Its linear range is broad that facilitates analysis of both large and small changes in promoter activity. Two luciferase reporter vectors containing either firefly or Renilla luciferase genes will co-transfect the cells. One reporter coupled to the GRP promoter will be normalized for the measurement while another coupled to a constitutive promoter will act as the internal control. The expression of the former relates to the changes on the GRP promoter at mRNA level while the latter minimizes the variability, including the differences in no. of cultured cells and the efficiency of cell transfection.

3. Materials and Methodology

3.1 pGL4 Vectors

Tang Wai Man (10525360D)

2012 Sem 2 ABCT316 Project Report

Figure 3.1 The maps of pGL4 vectors. (Source: Promega, Technical Manual of pGL4 Luciferase Reporter Vectors) Circle map of pGL4 vector Sequence reference points

Table 3.1 Comparisons of four pGL4 vectors from Promega [7].

pGL4 vector

Properties Contains luc2 reporter gene does not have promoter sequence

pGL4.10 [luc2] Contains luc2 reporter gene Has strong promoter, SV40 Contains luc2 reporter gene Has a minimal promoter Contains hRluc reporter gene Has SV40

pGL4.13 [luc2/SV40] pGL4.23 [luc2/minP] pGL4.74 [hRluc/TK]

Usage in this project For firefly luciferase assay As a -ve control if not recombinant with GRP promoter As target recombinant if having GRP promoter gene For firefly luciferase assay As a +ve control that strong luciferase activities are expected For firefly luciferase assay As +ve controls that low luciferase activity is expected For Renilla luciferase assay As a reference to minimize the inherent variability, like different cell no. and efficiency of transfection in different trials

3.2 Double Restriction Digestion Sticky ends of restriction enzymes allowed the easier ligation than blunt ends because it was no need to add linkers for further process after digestion. It was found that sticky ends could be created on pGL3-GRP78 by restriction enzymes either XhoI, SalI, BglII or BamHIthese were the same as on pGL4 vectorsin order to cut the gene from pGL3-GRP78 vector. It allowed the insertion of GRP78 to the XhoI or BglII sites upstream of luc+ or the SalI or BamHI sites downstream. GRP genes fitted between 76th base pair (bp) and 214th bp. XhoI and BglII cut at 74th and 214th bp respectively. Thus, XhoI and BglII would be used for double digestion of the vectors. Two restriction enzymes of different restriction sites prevented selfligation of digested plasmids itself.
Figure 3.2 Recognition sites of XhoI (left) (8) and BglII (right) [9]. (Sources: Promega)

3.3 Gel Red for DNA binding

3.4 The Wizard SV Gel and PCR Clean-Up System [10] This kit from Promega helped to extract DNA samples 100bp to 10kb from agarose gel after the running of the gel electrophoresis. This membrane-based system, which could bind to DNA, allowed up to 95% recovery of isolated DNA fragments within 20 minutes. 3.5 Cell Cultures The power form of high glucose Gibco DMEM (Dulbeccos Modifies Eagle Medium) [11] from Invitrogen was used for preparation of medium for mammalian cell culture. It contained high concentration of glucose (4.5g/L), amino acids and vitamins supporting fast growth of 293T cells. Also, powder form was cheaper than solution form. The phenol red present in DMEM indicated the pH of the medium.

Tang Wai Man (10525360D)

2012 Sem 2 ABCT316 Project Report

FBS (fetal bovine serum) [12] contained a mixture of essential factors for cell propagation. It could also act as a protective buffer against changes like changes of pH, heavy metals, proteolytic activity and endotoxin. FBS should be added to DMEM before feeding cells. DMEM with FBS should be used within one month because FBS would be degraded with time. LB (Lysogeny broth), a nutritionally rich medium, was used for supporting bacterial growth. PBS (phosphate buffered saline) is a water-based salt solution. It was used to wash away the waste and also DMEM medium that may affect the experimental result. The isotonic and nontoxic properties allowed the cells to maintain their structural and physiological integrity and it helped to maintain a constant pH during washing. Ampicillin was added into culture broth and plate to screen the colonies of transformed E.coli. Because pGL3 and pGL4 vectors contained ampicillin resistance gene already, only the transformed cells could grow in the medium theoretically. All materials in this part were sterile or filtered before use to prevent biological contamination. 3.6 T4 DNA Ligase for ligation

3.7 QIAprep Spin Miniprep Kit (QIAGEN) [] This kit was used to extract and purify the recombinant plasmid DNA from the E.coli. It was based on alkaline lysis of bacterial cells followed by adsorption of DNA onto silica in the presence of high salt. P2 buffer contained SDS to puncture holes in cellular membranes and was sued in conjunction with resuspension P1 Buffer of RNase to release DNA from cells and degrade RNA respectively. The silica membranes of the QIAprep columns could selective adsorbe plasmid DNA (while RNA, cellular proteins, and metabolites were found in the flowthrough) in high-salt buffer and elution in low-salt buffer. Buffer PB was used to wash and remove salts efficiently. Buffer N3 was for neutralization of the solution. Small elution buffer volume (50L of ddH2O) was used for elution to have high-quality and high-concentration plasmid DNA. The purified DNA was ready for immediate use and it was no need to precipitate, concentrate, or desalt. 3.8 Transient transfection Examples of popular transfection methods are microinjection, calcium phosphate, liposome, and retrovirus infection. The one of calcium phosphate will be used due to its effectiveness, lower cost, easy availability and easy handling. ProFection Mammalian Transfection System (17) (Promega, catalog no. E1200) will be used for the transfection. It is a formulation that is optimized for the maximal transfection efficiency, long-term stability and minimal cytotoxicity. It also provides a good performance in transient transfection of mammalian cells. 3.9 Dual-Luciferase Reporter Assay System (18) It assays the GRP promoter strength of vectors in transfected samples by comparing the luciferase activities (Promega, catalog no. E1910).
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4. Procedures
4.1 Restriction Digestion The restriction digestion was set up as the following table and incubated at 37 C. Afterward, the reaction Eppendorf tubes were stored at -20 C.
Table 4.1 Restriction digestion set up.
Plasmid DNA Plasmid stock concentration (g/mL) Amount needed (g) Restriction digestion Volume added in the Eppendorf (L) 1. ddH2O 2. Buffer (10X) 3. XhoI 4. BglII 5. Plasmid DNA Total volume (L) pGL3-GRP 624.5 4.0 Eppendorf 1 9.6 2.0 1.0 1.0 6.4 20.0 pGL4-10 198.5 1.0 Eppendorf 2 12.0 2.0 0.5 0.5 5.0 20.0

4.2 Preparation of LB Broth and Agar Plates LB broth medium (20g/L) were prepared by adding 10g LB powder into 200mL ddH2O first and mixed. The medium was marked up to 500mL with ddH2O to prevent the volume of LB powder affecting the total volume. The medium was autoclaved and stored at 4 until use. 500L of stock ampicillin (100g/L) was added into 500 mL of molten agar to have the required concentration of ampicillin (100g/mL). After swirling, the agar was poured to 20 agar plates (5 plates for each group and ~25mL agar for each plate). The plates were allowed to set in room temperature and be stored at 4 until use. 4.3 DNA Gel Electrophoresis Gel electrophoresis was performed to confirm the presence of the target bands after the restriction digestion. The 1% (w/v) agarose gel was prepared by adding 0.502g agarose powder into 50mL 1X TAE buffer. The agarose was mixed and melt in microwave oven. 5L of GelRed was added and then the gel was casted. The digested plasmids were spun down. After positioning the gel into the gel tank, 1X TAE buffer was used to fill the tank. The loading samples were prepared as table 2 and loaded into the gel wells. Then, the electrophoresis was performed at 100V until gel front reach 1/3 from the bottom of the gel. Required bands were identified and sliced under UV illumination. The slices were stored in pre-weighted Eppendorf tubes and the weights were determined.
Table 4.3 DNA gel electrophoresis set up.
Lanes from left to right Remarks Volume loaded (L) Gel weight of target band (g)

Tang Wai Man (10525360D) 1. 2. 3. 4. 5. 6. 100bp marker 1kb marker Digested pGL4-10 Digested pGL3-GRP Intact pGL4-10 (-ve control) Intact pGL3-GRP (-ve control) From Bio-Rad From Promega 20L DNA + 4L 6X loading dye 1L DNA + 4L ddH2O + 1L 6X loading dye

2012 Sem 2 ABCT316 Project Report 5 5 24 24 6 6

pGL4-10: 0.1538 GRP: 0.1649

4.4 Purifying Target DNA from Agarose gel The Wizard SV Gel and PCR clean up system were used. 154L and 165L of membrane binding solution were added to gel slices of pGL4 and GRP respectively. After the gel slices was completely dissolved in 65 water bath, the gel mixtures were transferred to SV Minicolumns that were inserted into collection tubes. After 1 minute of incubation at room temperature, the columns were centrifugated at 13000rpm for 1 minute. The flow-through was discarded and 700L of membrane wash solution with ethanol was added, following by another 1 minute of centrifugation at 13000rpm and discarded the flow-through again. This step was repeated with 500L membrane wash solution, and centrifugation for 5 minutes. After the discard of flow-through, further 1 minute for centrifugation with the lid opened was performed to allow the residual ethanol to be evaporated. Then, the Minicolumns were transferred to clean 1.5mL microcentrifuge tubes and 50L ddH2O was added. The tubes were incubated at room temperature for 1 minute, followed by the centrifugation at 13000rpm for 1 minute. The DNA obtained was stored at -20. 2L of each sample was used to measure the concentration of DNA after the gel purification by taking ddH2O as blank. 4.5 Ligation After calculated the required concentration of DNA obtained from previous purification for ligation (vector to insert weight ratio equal to 10:4 ng), the ligation reaction mixtures were prepared in the composition shown in table 3. The reaction was performed at room temperature for more than two hours and the tubes were stored at -20 until transformation.
Table 4.5 The ligation set up.
Ligation reaction 1. ddH2O (L) 2. Buffer (L) 3. Vector (pGL4-10) (L) 4. Insert (GRP) (L) 5. T4 DNA ligase (L) Total volume (L) (1) ligation product 3.5 1.0 2.0 2.0 1.5 10.0 (2) -ve control (no insert) 5.5 1.0 2.0 -1.5 10.0 (3) -ve control (no ligase) 5.0 1.0 2.0 2.0 -10.0

4.6 Preparation of Competent Cells The prepared diluted culture of E.coli cells of OD600 ~0.4 was spun at 4000 rpm for 10 minutes in 4. The medium was discarded and the pellet obtained was resuspended with 30mL ice-cooled 0.1M CaCl2. The cells were centrifugated at 4000 rpm for another 10

minutes in 4. The supernatant was discarded and the pellet was resuspended in 2 mL 0.1M CaCl2 with 15% glycerol. Finally, every 200L cell suspension was added into 11 Eppendorf tubes. The tubes were stored at -80 until transformation. 4.7 Transformation 5L of ligation product and controls (including ve control (no insert), -ve control (no ligase), intact pGL4-10, and ve control (no plasmid, i.e. ddH2O)) were added to the Eppendorf tubes of 200L E.coli cells separately. The tubes were then chilled at 4 for 10 minutes, followed by heat shock at 42 for 45 seconds. The tubes were then returned to 4 for 2 minutes. 800L liquid LB without ampicillin was added into the each tube and the tubes were incubated at 37 for 45 minutes with shaking at 220rpm. 50L of each tube was then spread on 5 agar plates with ampicillin. After the plates were dried, the plates were inverted and incubated at 37. 4.8 Clone Screening Two days after the transformation, 20L of 100 g/L ampicillin was added to 20mL LB broth. The LB medium of 100 g/ mL ampicillin was put into four 50mL centrifuge tubes, each 5mL. Four discrete colonies were picked from the transformation plate of pGL4-10-GRP plasmids and put into the centrifuge tubes separately. 4.9 DNA Extraction and Purification from Transformed E. coli By use of QIAprep Spin Miniprep Kit, the buffers were prepared according to the manufactures instruction for extraction of the amplified recombinant plasmid DNA from the E. coli culture plates. The pellets of competent cells were resuspended in 250L Buffer P1. 250L Buffer P2 was added to the Eppendorf tubes. The solution was mixed thoroughly and gently by inverting the tubes until a homogeneously coloured suspension was achieved. 350L Buffer N3 was added and the tubes were mixed immediately by inverting to avoid localized precipitation, followed by centrifugated at 13000rpm for 10 minutes. The supernatants collected were applied to the QIAprep spin column by decanting or pipetting, and centrifugated for 1 minute. The flow-through was discarded. Then, the columns were washed by adding 750L Buffer PE and centrifugating for 1 minute again. The flow-through was discarded, followed by centrifugation for 1 minute. The columns were placed in clean Eppendorf tubes. To elute the DNA, 50L ddH2O was added to the center of each column and the tubes were standed for 1 minute and centrifugated for 1 minute. The DNA concentrations and purities were determined and recored. Buffer P1 should be placed on ice to prevent degradation of RNase. Buffer P2 should be closed immediately to avoid acidification from CO2 in the air.

5. Results
5.1 Gel Electrophoresis of Restriction Digestion Products

Tang Wai Man (10525360D)

2012 Sem 2 ABCT316 Project Report

Figure 5.1 Results of DNA gel electrophoresis. Lane: (1) 100bp marker; (2) 1kb marker; (3) Digested pGL4-10; (4) Digested pGL3-GRP; (5) Intact pGL4-10 (-ve control); (6) Intact pGL3-GRP (-ve control). Dye front: 6.81cm.
1 0.9 0.8

Chart 5.1a Standard curve of 100bp DNA marker y = -0.4279x + 1.9005 R = 0.9869

1 0.8 0.6

Chart 5.1b Standard curve of 1kb DNA marker

Rf

0.7 0.6 0.5 2 2.5 3 3.5

0.4 0.2 0 2 2.5 3

y = -0.4143x + 1.8568 R = 0.9914

Rf

3.5

4.5

log(Molecular weight)

log(Molecular weight)

Table 5.1 The calculated molecular weight of each band found on the gel.
Lane 3 4 Band a b c 5 d Distance from origin 2.26 2.27 6.42 1.94 Rf 0.331865 0.333333 0.942731 0.284875 log(MW) calculated 3.650000 3.677207 2.238300 3.794170 Calculated bp 4466 4775 173 6225 Expected bp ~4200 ~4800 ~200 Comment Linear pGL4-10 Linear pGL3 Linear GRP Nicked / relaxed pGL4-10

e 6 f g h

3.00 1.52 2.14 2.95

0.440529 0.223201 0.314244 0.433186

3.418468 3.943034 3.723283 3.436190

2621 8771 5288 2730

Supercoiled pGL4-10 Relaxed pGL3-GRP Nicked pGL3-GRP Supercoiled pGL3-GRP

5.2 Concentration of Purified DNA

Table 5.2 The parameters of DNA obtained from purification of digested plasmids.
Parameters A A A A A
260 280 320 260 260

pGL4-10 0.400 0.291 1.118

GRP 0.252 0.262 0.165 0.897 0.212 4.4 50 220

/A

280 230

1.607 0.818 13.9 50 695

/A

DNA Concentration (ng / L) Volume obtained from purification (L) DNA obtained from purification (ng)

5.3 Transformation
Figure 5.3 The number of colonies resulted from transformation.

Tang Wai Man (10525360D)

2012 Sem 2 ABCT316 Project Report

Transformation efficiency =

11

5.4 DNA Concentrations and Purities of Amplified Recombinant Plasmids

Table 5.4 The pGL4-10-GRP DNA obtained from transformated E.coli.
Parameters A230 A A A A A
260 280 320 260 260

1 2.340 5.030 2.810 0.195

2 2.320 5.050 2.830 0.248 1.860 2.319 240

3 2.310 5.020 2.650 -0.043 1.881 2.153 253

4 2.050 4.350 2.160 -0.570 1.802 1.878 246

/A

280 230

1.847 2.251 242

/A

DNA Concentration (ng / L)

6. Discussion
6.1 Restriction Digestion of Plasmid DNA
Due to image problem, the dye front is assumed to be the bottom of the picture and the lane to be the top of the image. Moreover, the gel image is not captured in correct position that

cause wrong distance migration measured. Due to image contrast problem, the markers could not be easily observed. However, the image could be observed easily under UV box illumination. Gel was not setup in correct manner Samples were not run in straight line Thickness of the gel increased in top left hand corner position which caused uneven distribution of heat, which cause migration at a slower rate near the upper position. Highest: nicked / relaxed circular plasmid Middle: linear Lowest: supercoil Centifugation / extract for too long form linear / relax coil. Do not sure for correct cutting 13bp difference A260 : Relative high absorbance for nucleic acid A280: Relative high absorbance for protein A320: Turbidity of the solution, indicates possible contamination A260 / A280: If the value close to 0.6, it indicates protein contamination A260 / A230: Indicator for contamination by phenolic ion and other organic compounds Presence of ethanol in samples May be caused by the machine contamination or caused by scratching of pipette tips

Source of errors Bad practice Use the same pipette tip for measuring concentration of both pGL4-10 and GRP Use the same position of kimwipe to clean the sample site Calibrations of DNA samples are needed for each measurement For measuring GRP, the DNA concentration is 0.5 ng / L for ddH2O

6.2 Transformation

6.3

7. Reference
1. Little, E., M. Ramakrishnan, et al. (1994). "The glucose-regulated proteins (GRP78 and GRP94): functions, gene regulation, and applications." Crit Rev Eukaryot Gene Expr 4(1): 1-18.

Tang Wai Man (10525360D)

2012 Sem 2 ABCT316 Project Report

2. 3.

4. 5. 6.

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Banhegyi et.al. (2007). Endoplasmic Reticulum Stress. Ann. N.Y. Acad. Sci 1113:5871. H. Yoshida et al. (1998). Identification of the cis-Acting Endoplasmic Reticulum Stress Response Element Responsible for Transcriptional Induction of Mammalian Glucoseregulated Proteins. J. Biol Chem 273(50):33741-33749. 293T cells. GeneHunter Online. Available from: <http://www.genhunter.com/products/aptag-3/index.html> M.F. Carey et al. (2009). Transcriptional Regulation in Eukaryptes: Concept, Strategies, and Techniques. Cold Spring Harbor Laboratory Press. Bruce A. Sherfet. al. (1996). Dual-Luciferase Reporter Assay: An advanced CoReporter Technology Integrating Firefly and renilla Luciferase Assays. Promega Notes Magazine 57:2. (2006). Technical Manual of Dual Luciferase Reporter Assay System. Promega Corp. (2005). XhoI Usage Information. Promega Corp. (2008). Bgl II Usage Information. Promega Corp. (2010). Wizard SV Gel and PCR Clean-Up System Technical Bulletin. Promega Corp. Dulbecco's Modified Eagle Medium (D-MEM) powder (high glucose). Invitrogen. Fetal Bovine Serum. Allele Biotechnology. Rapid DNA Ligation Kit. Fermentas. Michelsen, B.K. (1995). Transformation of Escherichia coli increases 260-fold upon inactivation of T4 DNA ligase. Anal. Biochem. 225:172. TransformAid Bacterial Transformation Kit. Fermentas. (2003). Instruction Manual for PureLink HQ Mini Plasmid Purification Kit. Invitrogen. (2009). Technical Manual for ProFection Mammalian Transfection System. Promega Corp. (2011). Technical Manual for Dual-Luciferase Reporter Assay System. Promega Corp.

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