Techniques in Molecular Biology, Lab Report Example

Through the restriction digestion and ligation of the isolated Vibrio fischeri chDNA and PGEM-3Zf (+), the produced recombinant DNA molecules required to be transferred into a living organism for replication to produce clones (Winfrey et al., 1997). This study attempts to transform the competent E.coli DH5? cells with the mixture of religated molecules from different ligations with 1:1, 2:1, 3:1, and 4:1 insert to vector ratio set up in the previous study. The bioluminescent clones were expected from the religated molecule of the vector with the insert of lux operon of the V.fischeri. The second half of the study examined the average insert size of the clones of constructed V.fischeri genomic library and the orientation of the lux⁺  clone through restriction digestion with Sal I and restriction mapping with EcoR I, EcoR V, and Xba I restriction enzymes. The plasmid DNA free of chDNA of E.coli and cell debris required for the restriction mapping and analysis was prepared by modified rapid boiling mini-prep method (Holmes and Quigley, 1981; Winfrey et al., 1997).

For the desired bioluminescent clone of ­ lux operon, the conditions favorable were optimized by the unique selection of the cloning method, the enzyme for restriction digestion, vector and the appropriate host. The genomic library was constructed using shotgun cloning in which the genomic DNA was digested into random fragments and can be easily optimized to generate the larger and more desirable fragments in the case of lux operon which is 8.5 kb long (Jacobsson and Frykberg, 2001; Winfrey et al, 1997). The Sal I enzyme was specifically chosen to digest the insert as its high G+C content coupled with low G+C content of genomic DNA of V.fischeri results in the desired average fragment  length of 9 kb (Baldwin et al.,1990). The Pgem-3Zf(+) was used as vector  and the digested fragments of the insert and vector were ligated using T4 DNA ligase to incorporate the insert into the vector by setting up four different ligation reactions to optimize the chances of success in obtaining the lux operon clone (Dugaiczyk et al.,1975). The chosen bacterial host was bacterial plasmid—the vector (Mochizuki et al, 2006), and it was Escherichia coli DH5? which has fitting properties for host’s requirements.

The transformed cells were grown on LB-Amp-X-gal medium and screened for the glowing clones using blue/white screening. The blue/white screening involves the process of ?-complementation due to the specific properties of the vector, host and the agents used in growth medium. The applied PGEM -3Zf(+) is very small, high copy number plasmid which possesses the ?-lactamase gene conferring ampicillin resistance (Vieira and Messing, 1982). However, the E.coli does not possess such gene for ampicillin resistance and dies in an ampicillin medium (Goh and Good, 2008). Thus, the ampcillin resistance serves as substantial marker for the uptake of vector by allowing the cells having plasmid only to grow on the plates to form colonies (Vieira and Messing, 1982).

The X-gal used in the  medium is chromogenic substrate that changes its color when cleaved (Maas, 1999). The E.coli ­has lac operon containing the lacZ gene which encodes for the ?-galactosidase protein. This ?-galactosidase has the property of cleaving and hydrolyzing the colorless X-gal, changing its color to blue (Langley et al., 1975). However, the E.coli DH5? strain has the ?M15 mutation (?-fragment missing) in the lac­Z gene that results in the synthesis of the non-functional ?-galactosidase, thus inhibiting the change in color of X-gal (Langley et al., 1975). The PGEM-3Zf(+) contains a lacZ? gene and transcribes the missing ?-fragment of the non-functional ?-galactosidase of the E.coli, process known as ?-complementation. Thus, the E.coli DH5? strain containing the vector will produce functional ?-galactosidase when lac operon is introduced (Maas, 1999; Langley et al., 1975). The ?-complementation is a simple and rapid method of screening transformants. The PGEM -3Zf(+) has multiple cloning site (MCS) in the lacZ? gene which is the site of cut for the ­Sal I during digestion (Promega, Madison). If the plasmid do not have insert, it is probably ligated to itself, resulting in the intact lac­Z? gene. Thus, when the mutant E.coli take the vector without insert, the vector will transcribe the missing ?-fragment resulting in the functional ?-gal which cleaves X-gal into blue color. However, when the vector contains the insert, the insert gets ligated in the MCS site; the lacZ? gene is interrupted and the insert is transcribed rather than ?-fragment. Thus, the transformed bacterial cells which take up the vector with the insert, lack the ?-galactosidase activity due to the missing ?-fragment and there will be no change in color of X-gal. So, the colonies with and without the insert are white and blue respectively (Kawaguchi et al., 2008; Langley et al., 1975; Tolmachov, 2009; Winfrey et al., 1997).

Besides the ?M15 mutation, there are two other mutations that make E.coliDH5? the good host for the recombinant DNA. It has r_k mutant and lacks the restriction endonuclease activity which would have otherwise resulted in the digestion of the new vector being introduced (Matney et al., 1970). It is also recA mutant; if the host had a functional recA protein, it would allow the homologous recombination and insertion into the bacterial genome. This would prevent the DNA from being recovered during the mini-prep (Skarstad and Boye, 1988).

The lux operon occurs once in the genome of the V.fischeri and the probability to ensure its representation in the given library can be estimated statistically from the calculations based on the Poisson distribution (Clarke and Carbon, 1976). Mathematically, around 2200 clones would need to be screened to achieve 99% probability of the lux operon in the V.fischeri library based on the following equation: where I is the average size of the clones fragment (9 kb in this study) and G is the size of the target genome; V.fischeri of 4.25*10⁶ bp, N =2200 is the number of clones need to be screened and P is the desired probability (Sambrook et al., 1989). However, the use of Sal I and pGEM vector as mentioned earlier increases this probability of 1 out of 100 clones to be bioluminescent.

In the first trial, a total of 16 clones were screened but none of them was of glowing lux operon clone. The transformation went well with the maximum transformation efficiency of 6.1 x 10⁶ transformants/µg of pGEM added (Invitrogen Corporation, 2006). All the controls worked as expected. No colonies were observed for the negative control of TE buffer (Froger and Hall, 2007). For the positive control of uncut PGEM, 1220 blue colonies were observed with the maximum transformation efficiency indicating the intact vector which transcribed their missing ?-fragment to encode the functional ?-gal that converts the X-gal into blue color (Winfrey et al., 1997). Out of 16 clones, 12 were observed for 1:1 ligation and rest were observed for 2:1 ligation. No clones were observed for 3:1 and 4:1 ligation. Among the working ligations, the 1:1 ligation worked best with the maximum ligation efficiency (%white) of 12.5%. The third control of the digested PGEM has few blue colonies. This indicated that the Sal I worked efficiently during the digestion, which was further supported by the gel electrophoresis of the restriction digestion of pGEM and V.fischeri carried out to generate fragments for ligation. During digestion, the expected bright band of 3.2 kb was observed for the digested linear PGEM and a smear of fragments was observed for the digested chDNA with the greatest intensity at around 9 kb as expected (Promega Corporation, 2008; Winfrey et al., 1997; Engebrecht et al., 1983). The number of blue colonies was too high as compared to the clones (Dugaiczyk et al., 1975). The failure to get more clones and specifically the ­lux+ clone can be related to the less efficient ligation observed from the gel electrophoresis. The band of the digested linear pGEM was clearly visible in all the ligation products, indicating much of the vector has been left unligated. However, this band was the faintest for 1:1 ligation, contributing to the assumption that most of the vector has ligated in this case. Though there was no evidence of the vector ligating back on itself, but there was no upshift in the chDNA. The upshift in the chDNA was expected due the ligation of the vector to the chDNA (Pack et al., 2010). Generally, 1 out of 100 clones is expected to be lux⁺ clone, the chances of getting lux⁺ clone were meager as  number of clones counted were only 16 clones.

Since no lux⁺ clone was observed in the first trial, the transformation was carried out twice by using the leftover ligation. Though the maximum transformation efficiency was 5.96 x 10⁶ transformants/µg but overall it increased as compared to the first trial for the ligations (Invitrogen Corporation, 2008).

Though the whole ligations were used in the transformation, the whole genomic library was not screened. Mathematically, 2200 clones would need to be screened to achieve a 99% probability that all genes would be represented with the size of the V.fischeri genome (4.26×10⁶ bp) and the average fragment of the clone to be 9 kb as mentioned earlier. But, only 16 clones were screened which doubted the screening of the whole genome (Clarke and Carbon, 1976; Karlyshev et al., 1999).

The mini-prep was meant to recover the plasmid DNA free of the cell debris and the chDNA of the E.coli so that it can be used in further techniques of restriction mapping and digestion for the analysis of the size of the insert and the characteristics of the lux⁺ clone such as orientation (Tarczynski et al., 1994). Though the small scale miniprep does not produce highly purified plasmid due to RNA contamination, but it is very rapid method and can sufficiently isolate plasmid in the range of 1 to 5 µg (Winfrey et al, 1997). However, the mini-preps are not recommended when highly purified DNA is required such as cloning or when the plasmid is required in large amounts (Saha et al., 1989; Zhu et al., 2006; Winfrey et al., 1997).

Sal I was used for the restriction digestion to determine the average size of the insert. The observed average insert size of the lux^– clone was 6446 bp which is smaller and can be correlated with the smear of the fragments observed during the Sal I digestion from approximately 9kb to 4 kb (Winfrey et al., 1997). The insert size for the lux⁺ clone was mathematically calculated to be 7748 bp, but this fragment was clearly above the 8000 bp of the 1 kb ladder while looking on the gel.

For the restriction digestion, the controls of the uncut samples were run for the gel electrophoresis to compare the digested fragments with the original plasmid and to check if there was any concatamers or RNA contamination in the sample. By looking at the gel, it was observed that the bands for the clones were way higher than the band for the blue clone. This is due to the presence of large size insert in the clones along with the fragment of the vector. The blue pGEM clone and the lux⁺ clone showed smear around 700 bp and lower. This was due to the RNA contamination. The cut blue clone has fragment of 3480 bp due to the linear pGEM digested by Sal I and the presence of 1808  and 4174 bp in the undigested sample represented its supercoiled  and nicked form respectively. No band was observed for one of the sample of lux^– clone and even the bands were very lighter for the other samples. The low intensity of the bands corresponds to the less concentration of the DNA. No visible pellet of DNA was visible during mini-prep for the missing sample on the gel. The fragments of 11989 and 10366 bp for the undigested lux⁺ clone might be the concatamers as their intensity is very low as compared to the band of the real insert.

The restriction mapping by single, double and triple digestion with EcoR I, EcoR V, and Xba I was carried out to determine the orientation of the lux⁺ clone; if it is pUWL 500 or pUWL 501. It has two bacteriophage promoters (T7 and SP6) that can read the incoming insert from both end and results in two possible directions for the transcription of the insert; forward or reverse (Promega Corporation, 2008). The expected fragments for each orientation by the given enzymes were compared to the observed fragments from the digestion and it was determined that the given lux⁺ clone had pUWL500 orientation.

Both the EcoR I and EcoR V cut the plasmid at the single site, resulting in one fragment of 10351 bp mathematically. However, this fragment was estimated to be 12000 bp as expected because these fragments were above the range of the fragments of the ladder and cannot be extrapolated. The first discrepancy to determine the orientation was observed when compared the Xba I digestion fragments. Only one fragment of 9616 bp, but actually above the 10000 bp marker on the gel was observed which was comparable to the respective 11192 bp fragment of pUWL 500. Another fragment of 808 bp of pUWL50 was not missing; this could be due to the very small size and low concentration of the fragment. However, the double digestion with EcoR I and EcoR V further confirmed the 500 orientation as the 7709 and 5333 bp of the observed digestion were comparable to the 7395 and 4605 bp respectively. Even the EcoR I and Xba I supported the 500 orientation.

To begin with, the chDNA was isolated from the V.fischeri using the modified lysozyme/SDS procedure (Marmur, 1961) and was purified by phenol-chloroform extraction followed by precipitation with alcohol (Winfrey et al, 1997; Eickbush and Moudrianakis, 1978). The isolated and purified DNA was analyzed for quantity and purity using the UV-spectrophotometry by measuring the absorbencies at different wavelengths of 234, 260, 280 and 320 nm. The amount of the chDNA isolated was 1027 µg and was pure with the A₂₆₀/A₂₈₀ of 1.87. To generate the genomic library, the shotgun cloning was carried out in which the steps of restriction digestion, ligation of the digested fragments and the transformation of the competent cells with the ligation product were carried out sequentially. Both the vector [pGEM-3Zf(+)] and the chDNA were digested with the Sal I. On agarose gel electrophoresis, the partially digested chDNA appeared as a smear of fragments from around 23.1 kb to 4 kb and the digested PGEM appeared as a linear fragment of 3.2kb. The average size of the insert digested by the Sal I was expected to be 9 kb (Winfrey et al., 1997).

For replication of the recombinant DNA, the competent E.coli, specifically DH5? strain was used as it possesses characteristics of the good bacterial host for the plasmids as discussed earlier. The cells were transformed with the ligation reaction using heat shock method along with three controls of TE buffer, uncut PGEM and 1:10 dilution of the Sal I digested pGEM in TE buffer. The transformed cells were plated on the LB-Amp-X-gal medium and incubated overnight at 37?C for growth. Out of 18, 16 clones were observed for the 1:1 ligation reaction, indicating that the ligation with 1:1 insert to vector ratio worked the best. Overall, this study failed to get a clone of lux operon. The use of Sal I increased the average size of the insert size and the plasmid cannot take fragments over the size of 10 kb, thereby decreasing the number of clones to be screened. But even then it has been experimentally observed in the laboratory that at least 100 clones need to be screened to get a lux⁺ clone. With only 18 clones, the probability of lux⁺ clone was very meager and it proved true in this case.

To determine the average insert size of the clones and orientation of the manufactured lux⁺ clone, the plasmid DNA was recovered and isolated from the chromosomal DNA of the host.  One blue (pgem) clone, two different lux^– clones and three replicates of the manufactured lux⁺ clone were grown in LB-amp culture overnight for this purpose. The plasmid DNA recovered by this procedure was very low in concentration as no visible pellets were observed when precipitated with alcohol. Also, the intensity of the bands was very low; no visible band was seen for one of the plasmid. This might be due to the poor recovery of plasmids during miniprep. The samples were digested with Sal I to determine the average insert size of the lux^– clones. The agarose gel electrophoresis of the digestion gave the average insert size for the lux^– clone of 6446 bp; smaller than the expected. The insert size for the lux⁺ was approximately 9kb and the blue clone was positively the result of the PGEM ligated on itself as its two conformations ; nicked of 4.1 kb and supercoiled of 1.8 kb was observed for the undigested sample and the linear PGEM of 3.kb was also observed for digested sample.

In summary, the restriction mapping of the lux⁺ clone was carried out with EcoR I, EcoR V, and Xba I to determine the orientation if it was pUWL 500 or pUWL 501 and it was successfully determined that manufactured lux⁺  clone used in this study had pUWL 500 orientation. Since the small fragments were missing and there were errors in the calculations, estimations were made while constructing the restriction map.