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HONG KONG POLYTECHNIC UNIVERSITY

B. SC. (HONS.) APPLIED BIOLOGY WITH BIOTECHNOLOGY

ABCT316 Experimental Approach in Molecular Biology and Biochemistry

20112012 Semester 2

GRP Promoter ActivityAssay

Project Report

Tang Wai Man (10525360D)

Contents Page

1. Objectives 12. Background Information 23. Materials and Methodology 34. Procedures 75. Results 86. Discussion 87. Reference 9

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Tang Wai Man (10525360D) 2012 Sem 2 ABCT316 Project Report

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1. ObjectivesTo 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 Information2.1 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 ofthe 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 pGL3 pGL4

Generation Old NewReporter gene expression

Abnormal expression

Lower Higher 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.

Temporal response Slower Improved by destabilizing the luciferase genesUsability Lower HigherAs 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

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)

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2.3E. coli as Competent Cell

OD600: Number of viable cells does not exceed 108 cells / mL ensure that E. Colidoes 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. Itslinear 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 Methodology3.1 pGL4 Vectors

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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].

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pGL4 vector Properties Usage in this project

pGL4.10 [luc2]

Contains luc2 reporter genedoes not have promoter sequence For firefly luciferase assay As a -ve control if not recombinant with GRP

promoter

As target recombinant if having GRP promotergene

pGL4.13[luc2/SV40]

Contains luc2 reporter geneHas strong promoter, SV40 For firefly luciferase assay As a +ve control that strong luciferase activities

are expected

pGL4.23

[luc2/minP]

Contains luc2 reporter geneHas a minimal promoter For firefly luciferase assay As +ve controls that low luciferase activity is

expected

pGL4.74

[hRluc/TK]

Contains hRluc reporter geneHas SV40 ForRenilla luciferase assay As a reference to minimize the inherent

variability, like different cell no. and efficiencyof 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 eitherXhoI, SalI,BglII orBamHIthese

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

andBglII cut at 74th

and 214th

bp respectively. Thus,XhoI andBglII would be used for double

digestion of the vectors. Two restriction enzymes of different restriction sites prevented self-

ligation 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 afterthe 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.

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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 non-

toxic 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 transformedE.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 andwas 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 flow-

through) 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 theluciferase activities (Promega, catalog no. E1910).

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4. Procedures4.1 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 -20C.

Table 4.1 Restriction digestion set up.

Plasmid DNA pGL3-GRP pGL4-10

Plasmid stock concentration (g/mL) 624.5 198.5

Amount needed (g) 4.0 1.0

Restriction digestion Eppendorf 1 Eppendorf 2

Volume added in the Eppendorf (L)

1. ddH2O 9.6 12.02. Buffer (10X) 2.0 2.03. XhoI 1.0 0.54. BglII 1.0 0.55. Plasmid DNA 6.4 5.0

Total volume (L) 20.0 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 agarosepowder 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 RemarksVolume

loaded (L)

Gel weight of

target band (g)

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1. 100bp marker From Bio-Rad 52. 1kb marker From Promega 53. Digested pGL4-10 20L DNA

+ 4L 6X loading dye

24 pGL4-10: 0.1538

4. Digested pGL3-GRP 24 GRP: 0.16495. Intact pGL4-10 (-ve control) 1L DNA + 4L ddH2O

+ 1L 6X loading dye

6

6. Intact pGL3-GRP (-ve control) 6

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) ligation product (2) -ve control (no insert) (3) -ve control (no ligase)1.ddH2O (L) 3.5 5.5 5.02.Buffer (L) 1.0 1.0 1.03.Vector (pGL4-10) (L) 2.0 2.0 2.04.Insert (GRP) (L) 2.0 -- 2.05.T4 DNA ligase (L) 1.5 1.5 --

Total volume (L) 10.0 10.0 10.0

4.6 Preparation of Competent Cells

The prepared diluted culture ofE.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 with30mL ice-cooled 0.1M CaCl2. The cells were centrifugated at 4000 rpm for another 10

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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 (includingve control (no insert), -ve control (no ligase),

intact pGL4-10, andve control (no plasmid, i.e. ddH2O)) were added to the Eppendorf tubes

of 200LE.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 TransformedE. 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. Results5.1 Gel Electrophoresis of Restriction Digestion Products

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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.

Table 5.1 The calculated molecular weight of each band found on the gel.

Lane Band Distance

from origin

Rf log(MW)

calculated

Calculated

bp

Expected

bp

Comment

3 a 2.26 0.331865 3.650000 4466 ~4200 Linear pGL4-10

4 b 2.27 0.333333 3.677207 4775 ~4800 Linear pGL3

c 6.42 0.942731 2.238300 173 ~200 Linear GRP

5 d 1.94 0.284875 3.794170 6225 Nicked / relaxed pGL4-10

y = -0.4279x + 1.9005

R = 0.9869

0.5

0.6

0.7

0.8

0.9

1

2 2.5 3 3.5

Rf

log(Molecular weight)

Chart 5.1a Standard curve of 100bp

DNA marker

y = -0.4143x + 1.8568

R = 0.9914

0

0.2

0.4

0.6

0.8

1

2 2.5 3 3.5 4 4.5

Rf

log(Molecular weight)

Chart 5.1b Standard curve of 1kb

DNA marker

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e 3.00 0.440529 3.418468 2621 Supercoiled pGL4-10

6 f 1.52 0.223201 3.943034 8771 Relaxed pGL3-GRP

g 2.14 0.314244 3.723283 5288 Nicked pGL3-GRP

h 2.95 0.433186 3.436190 2730 Supercoiled pGL3-GRP

5.2 Concentration of Purified DNA

Table 5.2 The parameters of DNA obtained from purification of digested plasmids.

Parameters pGL4-10 GRP

A260

0.400 0.252

A280

0.291 0.262

A320 1.118 0.165A

260/

A

2801.607 0.897

A260

/ A230

0.818 0.212

DNA Concentration (ng / L) 13.9 4.4

Volume obtained from purification (L) 50 50

DNA obtained from purification (ng) 695 220

5.3 Transformation

Figure 5.3 The number of colonies resulted from transformation.

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11Transformation efficiency =

5.4 DNA Concentrations and Purities of Amplified Recombinant Plasmids

Table 5.4 The pGL4-10-GRP DNA obtained from transformated E.coli.

Parameters 1 2 3 4

A230 2.340 2.320 2.310 2.050

A260

5.030 5.050 5.020 4.350

A280

2.810 2.830 2.650 2.160

A320

0.195 0.248 -0.043 -0.570

A260

/

A280

1.847 1.860 1.881 1.802A

260/ A

230 2.251 2.319 2.153 1.878

DNA Concentration (ng / L) 242 240 253 246

6. Discussion6.1 Restriction Digestion of Plasmid DNA

Due to image problem, the dye front is assumed to be the bottom of the picture and the laneto be the top of the image. Moreover, the gel image is not captured in correct position that

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cause wrong distance migration measured.

Due to image contrast problem, the markers could not be easily observed. However, theimage 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 unevendistribution 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 measurementFor measuring GRP, the DNA concentration is 0.5 ng / L for ddH2O

6.2 Transformation

6.3

7. Reference1. 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.

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2. Banhegyi et.al. (2007). Endoplasmic Reticulum Stress. Ann. N.Y. Acad. Sci 1113:58-71.

3. H. Yoshida et al. (1998). Identification of the cis-Acting Endoplasmic Reticulum StressResponse Element Responsible for Transcriptional Induction of Mammalian Glucose-

regulated Proteins. J. Biol Chem273(50):33741-33749.

4. 293T cells. GeneHunter Online. Available from:

5. M.F. Carey et al. (2009). Transcriptional Regulation in Eukaryptes: Concept, Strategies,and Techniques. Cold Spring Harbor Laboratory Press.

6. Bruce A. Sherfet. al. (1996). Dual-Luciferase Reporter Assay: An advanced Co-Reporter Technology Integrating Firefly and renilla Luciferase Assays. Promega Notes

Magazine 57:2.

7. (2006). Technical Manual of Dual Luciferase Reporter Assay System. Promega Corp.8. (2005). XhoI Usage Information. Promega Corp.9. (2008). Bgl II Usage Information. Promega Corp.10. (2010). Wizard SV Gel and PCR Clean-Up System Technical Bulletin. Promega Corp.11. Dulbecco's Modified Eagle Medium (D-MEM) powder (high glucose). Invitrogen.12. Fetal Bovine Serum. Allele Biotechnology.13. Rapid DNA Ligation Kit. Fermentas.14. Michelsen, B.K. (1995). Transformation ofEscherichia coli increases 260-fold upon

inactivation of T4 DNA ligase. Anal. Biochem. 225:172.

15. TransformAid Bacterial Transformation Kit. Fermentas.16. (2003). Instruction Manual for PureLink HQ Mini Plasmid Purification Kit.

Invitrogen.17. (2009). Technical Manual for ProFection Mammalian Transfection System. Promega

Corp.

18. (2011). Technical Manual for Dual-Luciferase Reporter Assay System. PromegaCorp.

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