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to analyse the SAA2 5' flanking region. The pGL2-basic vector is 5597 kb in size, it

contains the cDNA encoding firefly luciferase, an ampicillin resistance gene for use in

screening of positive clones, an fl origin of replication derived from filamentous phage,

an origin o f replication in E.coli (ori) an SV40 region containing a poly(A) signal which

lowers background activity o f the promoter from spurious transcription of the reporter

gene. The vector contains a multi cloning site with 8 restriction enzyme sites for use in the

cloning of DNA fragments in to the reporter vector .

ooiyJA) signal

(lor b a c k g ro u n d red u ctio n ) A m o '

o o 'y (A i s ig n a l ('o r lu c r e o o n e i ') \

SV40

luc

Fig 3.2. The pGL2 luciferase reporter vector used to make SAA2 5' flanking region reporter constructs.

DNA fragments containing regulatory elements which are cloned into the polylinker drives

expression of the reporter gene, the cDNA for firefly luciferase. Luciferin is a organic acid

present in fireflies, it reacts with ATP to form the intermediate luciferyl-adenylate. In the

presence o f oxygen and magnesium the enzyme luciferase catalyses the oxidation o f the

intermediate producing oxy-luciferin, ADP and a photon of light (DeWet et al, 1987) (fig

3.3.). Relative levels of expression of the gene, due either to the vector promoter or

inserted DNA fragments, are determined by measuring the ability of the luciferase to

catalyse this reaction.

lucifcr3S0

luciferin + ATP luciferyl-AMP ---> ■ Oxy-luclferin + ADP

+ light

Fig. 3.3. The light emiting reaction which occurs in the firefly and is catalysed by the enzyme luciferase

Fragments of the SAA2 5' flanking region were used to make the reporter constructs (fig

3,4). The fragments were identical to those used in chloramphenicol acetyl transferase

(CAT) reporter constructs by Edbrooke and collègues previously in this laboratory

(Edbrooke etal, 1991). The Bam-luc construct was made by digestion o f the Bam-CAT

construct with BamHI and Bglll to remove the fragment from the CAT reporter

construct. The fragment was then ligated into the bglll site of the pGL2 polylinker region.

The Xmn-luc construct was made by digesting Bam-CAT with XmnI and Bglll and

cloning the isolated fragment into the Smal and Bglll sites of the pGL2-Basic vector. The

Sst-luc construct was made by digesting Sst-CAT with SstI and Bglll and cloning the

isolated fragment into the Smal and Bglll sites of the pGL2-Basic vector. The Hind-luc

from the -444 (Hindlll site) to the hindlll site of the multicloning site of the plasmid. The

fragment was cloned into the Hindlll site o f the pGL2-basic vector. The Sau-luc

construct was made by digestion o f the Sau-CAT construct (Edbrooke et al ) with Sail

BamHI Sail PstI Hrndm Bam-CAT/ Sst-CAT/ Sau-CAT/ BamHI BfQI

Ligate into Xhol/Bgin sitea of pC3JZ*baKic

Bam-luc

Sst-luc Xnm-hic

ligate into Ifiodni site of pCE.2-i>anc

ends of vector

Hind-luc

and Bgin to remove the fragm^t from the CATreporter vector, the fragment was cloned

into the Xhol and B glll sites o f the pGL2 basic vector. The Sma construct was made by

digestion o f the Sau-luc construct with Sma to remove the SAA2 5' flanking region 5' o f

the Smal site. The ends o f the construct from which the fragment was removed were

religated back together.

3.2.1. Ligation reactions

Digested DNA fragments and vectors were isolated by electrophoresis through a 1%

agarose gel, excision o f the required DNA band from the gel and subsequent removal from

the agarose using the Qiaex extraction system from Qiagen. Ligations were performed in

a reaction mix containing DNA as specified below, Ix ligase buffer (supplied with

enzyme) and 1 unit T4 DNA ligase. The reaction mix included 50 ng o f vector and

approximately ISOng o f insert, to give a insert to vector molar ratio o f approximately

5 .1. To religate the ends o f constructs o f which regions are to removed the reactions

contained approximately 50ng o f the construct to be religated. The ligation reactions, at

a total volume o f lOpl, were carried out at 14°C for approximately 16 hours.

3.2.2. Transformation of bacteria

The ligated D N A s were transformed into 'One Shot' competent cells bacterial strain

INVaF, considered to be equivalent to the strain DH5afrom invitrogen. p-

mercaptoethanol was added to the cells to a total concentration o f 0.02M and incubated

on ice for 2 mins. To a 2 0 pl aliquot o f the cells was added 5pl o f ligation reaction mix,

followed by incubation on ice for 30 mins. The cells were heat shocked at 42°C for 45

seconds and then incubated on ice for a further 2 mins. To the cells was added 400pl o f

SOC media ( see section 2.4.1.) and the cells were incubated at 37°C for 1 hour with

the plates incubated at 37°C overnight.

3.2.3. Colony PCR

Bacterial colonies from transformation plates were screened for positive clones by colony

PCR. Colonies were picked using a inoculating needle. The needle was then 'dipped' into