Design and Application of a Shuttle Vector System (II)

4.1. Introduction

Each o f the methods for the genesis o f recombinant viruses deseribed in the preceding sections suffered from the same problems - low efficiencies in the second-round PCR and in the digestion and ligation o f these PCR products into plasmid vectors, intra- bacterial re-arrangement o f plasmids, and poor, if any, amplification during ‘final’ PCRs. As a result, the shuttle vector was re-designed with a new cloning site to improve both ligation and PCR efficiencies.

In the previous chapter, the cloning site o f pHIVEC was itself cloned into a shuttle vector. However, when designing this cloning site, the list o f possible restriction sites was constrained to those absent from the full-length pHXB2ART molecule. Here, the cloning site o f pGEM-TART was re-designed using a broader range o f potential restriction sites. The original BstBl and SacW restriction sites were replaced by M fel and X b a \ sites respectively.

In addition to retaining maximal amounts o f sample-derived RT domains, this new vector (pRTshut) possesses two signifieant advantages over pGEM-TART. Firstly, ligation efficiency improves with longer sticky ends - M fe\ and X b a l cleave to leave four-base stieky ends as opposed to the two-base overhangs left by each o f BstBl, N arl, and SacW. Secondly, only single base mismatches between the primers and target sequences are required for their introduction during second-round PCR amplification, contrasting favourably with the multiple mismatches with the ‘Inner’ and ‘IN ’ primer sets (compare FIGURE 2-6 and 3-4 with FIGURE 4-7). Finally, the second-round antisense primer specifies less o f the 3 ’ end o f RT, allowing more sample RT sequence to be represented in each recombinant virus. As an additional measure, a third bacterial strain (STBL2, section A.3) was employed for its supposed suitability in maintaining

the integrity o f Po/-containing plasmids. Aside from the identities o f the restrietion sites, this protoeol is as per the previous shuttle vector system, illustrated in FIGURE 3-1, page 95

4.2. pRTshut

The set o f restriction enzymes lacking sites in either pGBM-TART or HIV-1 RT is much greater than that used in the design o f pHIVEC. This was due both to the shorter length o f pGEM-TART relative to pHIVEC, and to the multiple cloning site o f ancestral pGEM-T Easy Vector having been removed from pGEM-TART, Vector NTI software was used as deseribed in section 2.2 to locate potential unique restrietion sites which were silent with respect to the amino acid sequence o f HIV-1 P o l (FIGURE 4-1). At the 5 ’ end o f RT, it was found that an M fel site could be introduced across bases 11-16 (5 nucleotides downstream o f the 55^61 site). Similar mutagenesis scanning across the RT- integrase junction o f P ol revealed three potential sites - a BamH l site spanning bases 1,664-1,669 o f RT, a B seA l site across bases 1,667-1,672 o f RT, and an X bal site over bases 3-8 o f integrase. To introduce either o f the first two sites using second-round PCR primers would have necessitated an antisense primer specifying the nine 3 ’-terminal amino acids o f RT. Moreover, introduction o f the BamHl site into PCR products would have required two mismatched bases within the PCR primer, a strategy thought unlikely to improve on previous experience. Therefore, the X bal site was selected as it required an antisense primer with just one deliberate mismatch, specifying only the three 3 ’- terminal amino acids o f RT (FIGURE 4-2).

p I S P I E T

C C C A T T A GC C C T A T T GAG A C T 5 ’ terminus o f HXB2 RT

CA A T T G - M f e l

G I R K V L F L D

GGA A T C AGG A AA G TA C T A / T T T T T A G A T HXB2 RT-Integrase

GA A T T C - BamlAl T C T A G A - X b a l T C CGG A -B am X ll

FIGURE 4-1: HXB2 sequences at the 5' and 3' ends o f R T showing potential silent restriction sites f o r use in a new shuttle vector. M ismatches are highlighted in bold type.

BstBl Smal S acll

C C C A T |T T C G AA|t - T T G|CC CGG g|g t C T |C CG C G q G t f pGBM-TART

C C C A T T A GC C|CA A T T g||CC CGG g|gT C T |C CG C GG|G (^ pRTshut

Mfel Smal S acll

Integrase ►

<t)A A T C AGG AAA G TA C T A / T T T T T A G A T GGA <t)A A T C AGG AAA G T A C T A / T T T C T A G A T GGA

pGEM-TART pRTshut X bal

FIGURE 4-2: Comparison o f the nucleotide sequences and restriction maps o f the RT- deletion regions ofpGEM -TART an d pRTshut.

Two oligonucleotides were designed specifically for the construetion o f pRTshut. As they were not used elsewhere in this projeet, they are deseribed below in TABLE 4-1 and FIGURE 4-3. Two PCRs were performed on pGEM-TART to introduce the required restriction sites, one using the primer pair T7 and MUN-1, the other using INT- X and a-Sp6 (both T7 and a-Sp6 are detailed in TABLE A-1). To the former was added 5U o f both SacW and Apal, and to the latter 5U o f both .Sadi and £coR I (shortening the PCR products to 569bp and 438bp respectively). Reactions were incubated at 37°C for 2 hours. Three separate digests were performed on pGEM-TART as detailed in TABLE 4-2. INT-X ' * ^ ... HXB2 PR &9/BI |5 'a d l Smal T7 _613bp MUN-1 Mfel ■ .Sadi HXB2 IN ;

r

FIGURE 4-3: Schematic diagram o f the RT-deletion region o f pGEM-TART, showing the alignments o f the mutagenic primers IN T-X and MUN-1.

TABLE 4-1: Specific oligonucleotides used in the synthesis o f pRTshut.

INT-X GGG ATG T C T GAA CCG TAG CGG ATA

GAA TCA GGA AAG TAC TA T T T C TAG

M UN-I T T T TAA CCT TGG GAT GAA ACC AAT CGC T T A

A ll five digests were separated by agarose gel electrophoresis (FIGURE 4-4). The rearmost bands o f the pGEM-TART digests and the stronger o f the two bands in each o f the digested PCR lanes were excised and purified (see boxed bands in FIGURE 4-4). Three cross-ligations were performed - one containing the Apal-SacW-àigQsiQà backbone and PCR fragment, one containing the E'coRI-6'acII-digested backbone and PCR fragment, and one containing both digested PCR fragments and the A pal-E coR i- digested backbone. As before, these 20pl ligations had a 3:1 molar ratio o f PCR fragment(s) to backbone, contained T4 D N A ligase in the appropriate buffer as supplied by NEB, and were incubated overnight at 12°C before being transformed into TOPI OF’ E. coli (section A. 10).

Colonies arising from each ligation were screened using S-HR and AS-HR (394bp product), followed by digestion with the enzyme appropriate for the mutagenic primer used to generate the insert, i.e. X bal for those inserts derived from the INT-X-aSp6- primed PCR, and M fel for those amplified by the T7-MUN-1-primer pair. The PCR product from one colony from the ‘INT-X -aSp6’ ligation successfully digested with X bal, and amplicons from four colonies from the ‘T 7-M U N -1’ ligation were successfully digested with Mfel. The single colony from the first plate was labelled pGEM-TART-A6al; the four colonies from the second were labelled pGEM-TART- M fel-A through D. The attempted ‘triple ligation’ containing the Apal-EcoK[-à\gQsXQà. backbone and both digested PCR products produced colonies which amplified to give a 394bp product, but none o f these products contained the desired restriction sites.

Plasmid D N A was extracted from cultures expanded from the five colonies detailed above by QIAGEN Plasmid Mini Kits (section A .8.2). Each extract was subjected to two separate restriction digests, one with S acll and A pal, and the other with S acll and EcoRI. Each digest generated two bands, separated by gel electrophoresis. The smaller o f the two bands were excised and purified from the S acll-E coR i digest o f pGEM- TART-A5aI and the A pal-S acll digests o f the four pGEM-TART-A(^I constructs (FIGURE 4-5). Conversely, the larger o f the two bands were excised and purified from the A p a l-S acll digest o f pGEM-TART-AZ?aI, and the Sacll-EcoKi. digests o f the four pGEM -TART-A^I constructs (FIGURE 4-6).

TABLE 4-2: The three restriction endonuclease combinations used to digest pGEM- TART, and the predicted band sizes thereof.

Digest No. I II 111

Enzymes A p a \ - S a c ll E c o R i - S a c ll A p a l - E coK l

3 J 9 7 3,527 2,959

Expected band sizes (bp)

568 438 1,006 3,675 bp 2,323 bp 1,929 bp 1,371 bp 1,264 bp 702 bp 1 kb 800 bp 700 bp 600 bp 500 bp 400 bp

FIGURE 4-4: Restriction endonuclease digests o f pG EM -TART and two mutagenic

PCRs (see text). Lane I - pG EM -TART uncut (supercoiled plasmid). Lanes 2-4 -

digests l- l l l detailed in TABLE 4-2. Lane 5 - T7-MUN-1-primed PCR digested with

A pa/ and Sac//. Lane 6 - INT-X-aSp6-primed PCR digested with S ac// and EcoRl. The boxed bands were excised and purified.

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