Subcloning of the PstI fragment from pMB into pUC19

1. introduction

      DNA should never be treated of considered as a static molecule since those molecules have a significant dynamic movement. It is constantly changing, and one mechanism by which this change is brought about is recombination.

     Recombination is the ability to transfer a specific gene from one organism to another resulting in offspring differ from either parent. It is a DNA re-arrangement between

two molecules of DNA having homologous sequences. The main objective of doing recombinant DNA technology is often to create a beneficial product or commercial ones.

      Recombination provides the tools through which genetics information is reformed. This, of course, provides an evolutionary advantage to eliminate the unwanted

mutations and to maintain the spreading of the desired mutations. Moreover, it has a significant role in conferring each individual a unique group of genetic information. Recombination must be taken place between precisely corresponding sequences to ensure no loss or addition for any base pair.

After the recombination has been done, in order to identify the recombinant vector from non-recombinant ones, two techniques must be used (Bolivar et al, 1977). First, growing the E. coli on the antibiotic-containing medium, which contains ampicillin. This one used to distinguish the recombinant bacteria from the non-recombinants. That can be determined easily since recombinant bacteria carry plasmid that has the ampicillin-resistance gene (AmpR), and in which can grow on ampicillin based medium, however, the non-recombinants cannot grow on the growth media since they have no resistance to the ampicillin (Smalla, Jechalke and Top, 2015). The problem, not all the plasmids transformed into cells may contain the inserted gene. Some of the cells do have the plasmid but without the inserted gene.

The second technique is the LacZ a-complementation (Fig1), which is a screening technique that allows the detection and distinguishes recombinants bacteria with insert DNA fragment from the one with only the plasmid but without the inserted gene by using blue/white selection to identify recombinant plasmid clones (Tolmachov, 2009).

 β-galactosidase is a protein produced by E. coli strain that carries the lacZ gene. In its active state, if bacteria are grown on an agar plate that has a substrate known as X-gal. when β-galactosidase is produced, X-gal is hydrolyzed(Tolmachov, 2009). Consequently, they produce an insoluble blue pigment. However, when Insertion of foreign DNA from pMB into the plasmid at the MCS region, which is located within the lacZα sequence (which can be cut by restriction enzymes),that would cause insertional inactivation(Smalla, Jechalke and Top, 2015). That would create a non-functional β-galactosidase enzyme, 

thereby it will stop the gene that produces α-peptide from working. Consequently, in cells that contain the plasmid with an insert in LacZα sequence, no functional β-galactosidase will form (Smalla, Jechalke and Top, 2015).

Therefore, The E.coli that has plasmid with no insertion appears blue in color since LacZ is functioning while the recombinant ones with insertion foreign DNA appear white. Consequently, it’s easy to pick the desired recombinant colonies from the culture.

 

 

 

 

 

 

1.1 Aim of the experiment

Subcloning experiment: transfer a DNA fragment from pMB to Puc19. Then detect the bacterial clones carrying the recombinant plasmid by using the insertional activation system.  Lastly, successful subcloning demonstrated by using restriction digest to confirm that the bacterial clones do contain recombinant plasmids.

2. Results:

Ampicillin

Kanamycin

Tetracycline

DH5

–

–

–

pUC19

+

–

+

pMA

–

+

+

pMB

+

–

–

XL1-blue

–

–

+

In this experiment, five different bacterial samples (Table1) have been tested for antibiotic resistance against three different antibiotics (Ampicillin, Kanamycin, Tetracycline). They are commonly used as selective agents in various cloning vectors.

       For the first antibiotic Ampicillin, only bacterial who contain Puc19 plasmid and pMB were resistant to Ampicillin, and the rest showed no growth. these suggest that pUC19 and pMB have the antibiotic-resistant gene in their plasmid (AmpR) against Ampicillin.

       However, for the Kanamycin, only bacterial that contain pMA plasmid were able to grow; these suggest that pMA plasmid has Kanamycin resistant gene.

       Lastly, for the Tetracycline antibiotic, bacterial who contain Puc19, pMA, and XL1-blue plasmids were able to grow. these suggest that bacteria that contain Puc19, pMA and XL1-blue plasmids have the antibiotic-resistant gene for Tetracycline

 

 

 

 

 

 

 

 

2.2. The double digests of pMA and pMB:

 

*Because of poor gel preparation (overheated and that increased the concentration of the gel) gel image has been obtained from group 4, everything below will be explained based on the date that has been obtained by group 4.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FIGURE  3. Restriction endonuclease digestions and electrophoretic separation of fragments of pMA and pMB

with a ruler to indicate the precise size of the fragment in centimeter. (refer to Fig 2, for the legend, since they share the same legend)

 on the left side, as marked from lane 1-7, it shows endonuclease digestions and electrophoretic separation of fragments of the plasmid pMA. On the right side as marked from lane 8-14 it shows endonuclease digestions and electrophoretic separation of fragments of the plasmid pMB. those abbreviation has been used to identify each restriction endonuclease enzyme as following

B: BamHI, E: EcoRI, P: PstI, X: XhoI and M: DNA marker

 

 

 

 

*cm

Bands

(lengths of the bands in kilo base pairs.)

2.65

10 kbp

2.75

8 kbp

2.9

6 kbp

3.09

5 kbp

3.2

4 kbp

3.38

3.5 kbp

3.59

3 kbp

3.72

2.5 kbp

4

2 kbp

4.5

1.5 kbp

5.25

1 kbp

5.88

0.75 kbp

6.7

0.5 kbp

7.8

0.25 kbp

FIGURE 4. measurement of DNA marker with a ruler. the y-axis represent the DNA marker measurement, while x-axis represent the ruler measurement in centimeter.

 

 

 

 

 

 

 

 

 

 

2.3. Restriction maps of pMA and pMB.

Table 3. Shows what has been electrophoresed on each lane from lane 8-14 (Fig 2) and what Restriction enzymes had been used on pMB plasmid, and the Size of the fragment that has been obtained. the numbers denote the lengths of the digestion products (fragments) in base pairs.

Table 2. Shows what has been electrophoresed on each lane from lane 1-7 (Fig 2.) and what Restriction enzymes had been used on pMA plasmid, and the Size of the fragment that has been obtained. the numbers denote the lengths of the digestion products (fragments) in base pairs.

 

lane

Digest performed on PMB

Size of fragment obtained

8

EcoRI +  XhoI

3100 bp, 1900 bp

9

EcoRI + PstI

3000 bp, 1300 bp, 790 bp

10

EcoRI + BamHI

4700 bp, 350 bp

11

BamHI + XhoI

2800 bp, 2200 bp

12

BamHI + PstI

2550 bp, 1275 bp, 1150 bp

13

PstI + XhoI

3600 bp, 1100 bp

14

XhoI

5050 bp

lane

Digest performed on PMA

Size of fragment obtained

1

BamHI

3600 bp

2

EcoRI +  XhoI

3600bp

3

EcoRI + PstI

2800 bp, 700 bp

4

EcoRI + BamHI

3000 bp, 400 bp

5

BamHI +  XhoI

3600 bp

6

BamHI + PstI

2400 bp, 1100 pb

7

PstI +  XhoI

3600 pb

 

FIGURE 5 Restriction endonuclease map for the digestion products presented in Table2. For pMA plasmid. The circular DNA has 3,600 bp (3.6 kb). With the EcoRI site arbitrarily placed at 12 o’clock position, the locations of the other mapped restriction endonuclease sites are marked.

FIGURE 6 Restriction endonuclease map for the digestion products presented in (Table3). For pMB plasmid. The circular DNA has 5,050 bp (5.05kb). With the EcoRI site arbitrarily placed at 12 o’clock position, the locations of the other mapped restriction endonuclease sites are marked.

The process of making restriction maps for circular DNA plasmid is, in general, the same as making a restriction maps for a liner DNA, except that each time that the restriction enzyme cleave produces a fragment. In other words, three fragments are formed when three sites are cut by the endonuclease enzyme, and so on. It is also important to mention that Each restriction enzyme cut at a specific site.

With the data obtained for pMA (Table 2),the deduction of a restriction digest map for the pMA plasmid is as follows.

       The source DNA plasmid is a 3.6 kilobase-pair (kb) produced with a single digest of BamHI, which suggests the length of the plasmid is 3.6 kilobase-pair since only one fragment has been obtained when the plasmid treated with single digest endonuclease.

        Digestion with (EcoRI + XhoI), (PstI + XhoI) or (BamHI + XhoI) produces a single restriction fragment, that suggests that there is no restriction site for XhoI, in other words, XhoI doesn’t cut pMA plasmid since there is no restriction site for XhoI.

       However, when double digest performed with (EcoRI + PstI), (EcoRI + BamHI) and (BamHI + PstI) produced two fragments. which suggests that EcoRI, PstI, BamHI each one of them has only one restriction site on the plasmid.

       The results of the EcoRI and PstI double digestion produced two fragments 2.8-kb, 700-bp. for the double digestion of EcoRI + BamHI produced 3-kb, 400 bp. for BamHI + PstI two fragments 2.4-kb, 1.1-kb produced. Based on that

 In general, the double digest of each of (EcoRI + PstI), (EcoRI + BamHI) and (BamHI + PstI) separately produced length, which roughly resonates with a length of the pMA plasmid.

 

With the data obtained for pMB (Table 3),the deduction of a restriction digest map for the pMB plasmid is as follows.

       The first single digestion (Fig 3/Table 3, lane 14) with XhoI produced one fragment, which is about 5.05-kb. The suggest the length of plasmid pMB is 5.05-kb.

       Digestion with XhoI and EcoRI (Fig 3/Table 3, lane 8), XhoI cleaves 5.05-kb EcoRI into two fragments 3.1-kb and 1.9-kb.

      Also, BamHI + XhoI (Fig 3/Table 3, lane 11), the double digestion of BamHI + XhoI, produced two fragments. That indicates XhoI, BamHI, and EcoRI cut at one site, and each one separately produces one single fragment.

      However, for the PstI restriction enzyme, when double digest performed with EcoRI (Fig 3/Table 3, lane 9), three fragments have been produced 3-kb, 1.3kb, and 790 bp. Moreover, when double digested with PstI + BamHI (Fig 3/Table 3, lane 12),three fragments also have been generated with different 2.55-kb 1.275-kb and 1.15-kb. Based on that, the date obtained for the gel electrophoresis image (Fig3) suggests that pstI cuts in two sites on the pMB plasmid. pstI has two recognized restriction sites inside the plasmid.

That been said, however, when double digests performed for pstI + XhoI  (Fig 3/Table 3, lane 13) only produced two fragments, 3.6-kb and 1.1-kb.

 

 

The similarities and differences between the two plasmids.

 

After the double digest and based on the data, the following similarities and differences between pMA and pMB plasmid have been observed:

First, the length, based on the restriction maps obtained from the gel electrophoresis (Table 3, Fig 3) pMA, is shorter than pMB (Fig 5) since pMA is about 3.6-kb whilst pMB is about 5.05-kb. That resonates with () that pMB derived from pMA.

Second the restriction site,

As shown and indicate in the restriction map above (Fig 5), XhoI doesn’t cut pMA plasmid, which indicates there is no restriction site for XhoI in pMA plasmid. However, with pMB, XhoI produce a single fragment during endonuclease (Fig 6/ Table 3)

For the PstI enzyme, based on the result that has been obtained, PstI has one site when digest pMA plasmid and produced only one fragment, which indicates that it has only one restriction site on the pMA plasmid. However, when pMB double digested PstI with EcoRI or BamHI, it produced three fragments, and since  EcoRI and BamHI cut only once based on the data ( fig 4,5 table 4,5) that indicates and suggest that PstI has two restriction sites in pMB plasmid.

3. DISCUSSION

For some restriction endonuclease mapping experiments, the sum of the fragments of some multiple digestions is less than the total length of the starting DNA because the fortuitous locations of some sites produce fragments of the same size. Under these conditions, two different fragments

with the same length that migrate to the same location in a gel after electrophoresis

often stain more heavily than a band with only one kind of fragment.

This difference in staining intensity gives an indication that

coincidental fragments have been produced by restriction endonuclease

digestion.

The resolution of fragments for restriction endonuclease mapping can

be enhanced by labeling the pieces of DNA, usually at the 5’ends, with a

radioactive compound or fluorescent dye and determining their lengths

after electrophoretic separation with autoradiography or fluorography,

respectively. A standard 5’-end-labeling procedure entails dephosphorylation

of the 5′ends of a linear DNA molecule with calf intestine alkaline

 

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