Abstract
Molecular cloning is a method to produce quantities of a specific DNA segment. It contains an abundance of techniques including DNA transfer, DNA recombination, DNA sequencing and so on. Though this practical, restriction maps were draw for plasmid pMA and pMB by doing single and double digest, a pMB fragment digested with PstI was inserted to plasmid pUC19 and then transferred to host cells to have proliferation and expression, and the sequence of PstI digested pMB fragment was analysed.
1. Introduction
Recombinant DNA molecules are molecules containing DNA sequences derived from more than one source. In molecular cloning, by using recombinant DNA, a specific combination of genes can be put into a carrier, and then can be proliferated and expressed in a recipient cell.
In medicine, by making use of molecular cloning, scientists have successfully constructed engineering strains of insulin, growth hormone of human, cattle and chicken, human interferon, erythropoietin, antigen of hepatitis B virus and antigen of foot-and-mouth disease virus, and conducted a large-scale production by fermentation industry. In gene therapy, there is a possibility of reversing cancer cells to normal cells through genetic engineering, for example, mouse tumor cells caused by SV40 virus can reverse to normal cells at high temperature.
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Many chemical reagents such as acrylic acid, ethylene glycol, methanol, ethylene oxide and salicylic acid can possibly be produced by making use of molecular cloning. In environmental protection, people transfer genes of one microorganism into another through genetic manipulation to create new strains that are more capable of degrading harmful substances, in order to break down toxic substances in industrial waste.[1,2]
Blue-White selection is a method for screening recombinant DNA. Vectors containing a β-galactosidase gene (lacZ) can have a complementation (α-complementation) with E.coli strain to form a functional β-galactosidase enzyme. Neither vectors, nor host cells have the enzyme activity. The lacZ gene has an internal multiple cloning sites (MCS) which can be cut by different restriction enzymes. Therefore, when a gene fragment is inserted in the vector, the lacZ gene will be disrupted and cannot form active β-galactosidase enzyme. X-gal can be metabolized by β-galactosidase to gain a blue product. Therefore, in the presence of X-gal, DNA with no insert gene can display a blue colour, while recombinant DNA, which have no enzyme function, display a white colour.[3]
The aim of the practical is to draw restriction maps of simple plasmids for recombinant DNA, do basic molecular cloning and sequence a DNA fragment.
2. Results
Table 1: Antibiotic resistances of 5 E. coli strains
LB/Ampicillin
LB/Tetracycline
LB/Kanamycin
DH5a
No growth
No growth
No growth
pUC19
Grown
No growth
No growth
pMA
Grown
Grown
No growth
pMB
No growth
Grown
Grown
XL1-Blue
No growth
Grown
No growth
DH5a: E. coli strain DH5a; pUC19: E. coli strain DH5a containing plasmid pUC19; pMA: E. coli strain DH5a containing plasmid pMA; pMB: E. coli strain DH5a containing plasmid pMB; XL1-Blue: E. coli strain XL1-Blue.
NO.
DNA
Enzyme
1
pMA
Bam HI
2
pMA
XhoI
3
pMA
PstI
4
pMA
EcoRI
5
pMB
Bam HI
6
pMB
XhoI
7
pMB
PstI
8
pMB
EcoRI
9
Lambda marker
10
ϕX174 marker
NO.
DNA
Enzymes
1
pMA
EcoRI, Bam HI
2
pMA
EcoRI, PstI
3
pMA
EcoRI, XhoI
4
pMA
Bam HI, PstI
5
pMA
Bam HI, XhoI
6
pMA
PstI, XhoI
7
pMB
EcoRI, Bam HI
8
pMB
EcoRI, PstI
9
pMB
EcoRI, XhoI
10
pMB
Bam HI, PstI
11
pMB
Bam HI, XhoI
12
pMB
PstI, XhoI
13
Lambda marker
14
ϕX174 marker
1
Lambda marker
2
Blue colony digested with PstI
3-7
White colonies digested with PstI
8
ϕX174 marker
gagtantagttcgccngttaatagtttgcgcaacgttgttgccattgctgcaggggggggggggaaagccacgttgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgaacaataaaactgtctgcttacataaacagtaatacaaggggtgttatgagccatattcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgatgaggttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttttaatgagaattggttaattggttgtaacactggcagagcattacgctgacttgacgggacggcggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctcaacgactgagtatggaccttcttcacgaggcagacctcagcgccccccccccccctgcaggca
Enzyme
No. of cuts
Position of sites (bp)
Recognition sequence
PstI
2
52, 1243
ctgca/g
XhoI
1
204
c/tcgag
E X Stop F A X Stop _ F A Q R C C H C C R G G G E S H V V S Q N L Stop C Y I A Q D K N I S S Stop T I K L S A Y I N S N T R G V M S H I Q R E T S C S R P R L N S N M D A D L Y G Y K W A R D N V G Q S G A T I Y R L Y G K P D A P E L F L K H G K G S V A N D V T D E M V R L N W L T E F M P L P T I K H F I R T P D D A W L L T T A I P G K T A F Q V L E E Y P D S G E N I V D A L A V F L R R L H S I P V C N C P F N S D R V F R L A Q A Q S R M N N G L V D A S D F D D E R N G W P V E Q V W K E M H K L L P F S P D S V V T H G D F S L D E V I F D E G K L I G C I D V G R V G I A D R Y Q D L A F S K I W Y Stop _ S Stop Y E Stop I A V S F D A R Stop V F L M R I G Stop L V V T L A E H Y A D L T G R R L C Stop I N R T F A E L K D Q I T H L P D N A D R S V A K Q K F K I T N W S T Y N K A L I N R G S L T F W L D D G A I Q A S T T E Y G P S S R G R P Q R P P P P C R
Aminoglycoside 3′-phosphotransferase, putative
3. Discussion
3.1 Antibiotics resistances
Seen from table 1, DH5a has no resistance to any of the three bacteria, pUC19 is resistant to ampicillin, pMA is resistant to ampicillin and tetracycline, pMB is resistant to tetracycline and Kanamycin, and XL1-Blue is resistant to tetracycline.
Plasmid pUC19, pMA and pMB, which were used in the cloning procedure, had different antibiotic resistances, while the bacterial host, DH5a, have no antibiotic resistance. Therefore, cells containing recombinant DNA could be selected by growing host cells in presence of antibiotic. Even when different plasmids are contained in the host cells, this method can be used. For example, tetracycline can be used to select cells containing only pMA from a mixture of cells containing pMA and pUC19.
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3.2 Restriction maps and relationship of pMA and pMB
From the single digest (1), pMA could be cut by Bam HI, PstI and EcoRI, and each enzyme could cut pMA once. However, pMA could not be cut by XhoI. pMB could be cut by Bam HI, XhoI and EcoRI once, and cut by PstI twice. Therefore, pMA has three restriction enzyme sites, while pMB has five. From the double digest (2), the results were consistent with single digest, and the length of each fragment could be obtained.
Restriction maps (3) were drawn based on the single and double digests. From the restriction maps, the fragments in pMA and pMB, cutting by Bam HI and EcoRI, have the same base pairs (430bp). The fragment cutting by EcoRI and PstI in pMA has the same base pair (720bp) with one of the fragments cutting by EcoRI and PstI in pMB. The fragment cutting by Bam HI and PstI in pMA has the same base pair (1150bp) with one of the fragments cutting by EcoRI and PstI in pMB. The longer fragment in pMB cutting by PstI was round about 3780bp, which was very close to the length of pMA (3800bp).
As all the lengths of fragments were roughly obtained and were not accurate. Therefore, we can assume that pMA is a part of pMB. pMB can be cut by PstI. If the longer fragment is re-circled, it will have the same base pairs and restriction enzyme sites (PstI, EcoRI and Bam HI) with pMA. The XhoI restriction site on pMB is between the two restriction sites of PstI, therefore, the longer fragment cannot be cut by XhoI, which is consistent with pMA. Seen from the antibody resistances, pMA is resistant to ampicillin and tetracycline, pMB is resistant to tetracycline and Kanamycin. This might because the tetracycline resistant gene is in pMA, which is a part of pMB. And kanamycin resistant gene is in the PstI fragment of pMB, which pMA does not have. For the ampicillin resistant gene, it might be located around the PstI restriction site in pMA, which will be insertion inactive when insert the PstI fragment to pMA to make it become pMB, therefore, pMB does not have ampicillin resistance.
This hypothesis can be proved by sequencing pMA and pMB fragment cutting by PstI, which was not included in this experiment.
3.3 Sub cloning recombinant clones
In 4, the blue colony had only one band, which meant that there was only one PstI restriction site in the plasmid. This was consistent with pUC19 that did not have an insert fragment.
Four of the white colonies had two bands each, including one band located around 1200bp. These were the recombinant DNA, with the pMB fragment digested with PstI. One white colony (No. 7) did not have a band located around 1200bp, but a fragment shorter than that. This was also a recombinant DNA, with other fragment rather than PstI fragment. This might be caused by some impurities through the procedure.
3.4 Sequence analysis
The sequence of the PstI fragment in pMB was obtained by overlapping two fragments (forward and reverse). Seen from 5, there are two PstI restriction sites (ctgca/g) and one XhoI restriction site (c/tcgag), and the XhoI restriction site is between the two PstI restriction sites. Therefore, if the fragment is digested with PstI and XhoI, two fragments (152bp, 1039bp). This is roughly consistent with the restriction map of pMB which was not accurate.
The amino acid sequence shown in 6 is one of the six possible sequences (5’3′ Frame 1), methionine, which is a start of protein sequence, and stop codons are over striking. One potentially matching sequence of protein encoded in the PstI fragment of pMB shown in 7, aminoglycoside 3′-phosphotransferase, begins with the first methionine in the fragment and have a length of 253 amino acids.
4. Conclusion
This practical provide us a better understanding of how to make a recombinant DNA and molecular cloning technique. These experiences can act as fundament of further researches such as researches in cancer cells.
References:
[1] Williams Wu, Michael J. Welsh, [et al.] (2003) Gene Biotechnology (2nd edition).
[2] Gerald Karp. (2002) Cell and Molecular Biology (3rd edition).
[3] Benjamin Lewin. (2004) Gene Ⅷ (International edition).
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