Example Report

Warning : copying any part of this student example report is plagiarism

Recombinant DNA Technology: A student’s first practical application using the Escherichia coli host, the pBluscript IISK+ cloning vector, and the bacteriophage lambda insert.

Department of Biochemistry, University of Sydney, Sydney, Australia.

show/hide

photoJill gives her overall opinion of the report (24s):

photoSarah describes how she went about writing this report (1m):

The primary aim of this study was for students to learn and practice some baseline molecular biology techniques for synthetic gene construction and product analysis. Basic tools of recombinant DNA technology include Escherichia coli host cells, restriction enzymes and cloning vectors. Each of these is used in this experiment to clone the DNA of bacteriophage λ. Firstly, the λ DNA was digested with EcoRI to yield six fragments. Four of these fragments were candidates for insertion into the EcoRI restriction site of the pBluescript IISK+ phagemid cloning vector and transformation into the E. coli DH5 strain host. Students were provided with colonies from just one of these recombined products and given the task of identifying the size and orientation of the insert. This was done using alkaline lysis method of plasmid DNA isolation followed by restriction digest and gel elecrophoresis of it. The insert size and orientation was examined by estimating the fragment sizes using a reference standard. And comparing them with restriction maps of all possible insert configurations. The initial gel was not of a quality sufficient to resolve the fragment size and orientation. Therefore, a model gel is preferentially used for the analysis. Not only did these data reveal the size and orientation of the insert, but they demonstrated some interesting aspects of gel interpretation. This includes the phenomenon of ghost banding as well as the presence of nicked/relaxed constructs in the gel matrix. Finally, the productivity of the cloning system was examined via spectrophotometric analyses of the plasmid DNA. Keywords: pBluescript IISK+; bacteriophage lambda; Escherichia coli DH5; restriction analysis; non-directional cloning.

show/hide

photoJill comments on the introduction (45s):

photoSarah describes how she did the research for this report (25s):

Introduction

The in vitro production of proteins continues to burgeon in response to demand for their use in both small-scale exploratory research as well as large-scale therapeutic procedures [1]. Recombinant DNA technology is the platform for protein preparation. Its application requires experience in a number of baseline molecular biology techniques. This includes the definitive screening and identification of recombined DNA. To this end, the primary aim of this study was for students to learn and practice some of these useful skills.

Among the traditional and perpetually valuable tools in synthetic gene construction are the Escherichia coli host cell, restriction enzymes and phagemid cloning vectors. Each of these is used in this experiment. ...

The bacteriophage λ contains, five BamHI and five EcoRI sites. It yields six fragments when digested with the latter. The four internal fragments are suitable for insertion at the EcoRI site of the cloning vector pBluescript IISK+ (Figure 1). ....

The pBluescript IISK+ circular phagemid (2961bp) contains a polylinker with 21 unique restriction enzyme sites, T7 and T3 RNA polymerase promoters, and two selectable markers; resistance for ampicillin and the gene for β-galactosidase [2]. ....

After synthesis, transformation and cloning of the constructs, it is standard procedure for scientists to analyse their quality and composition. This is to: 1) confirm that the amplicon is the sequence of interest; and, 2) assist in optimizing the expression system based on its relative productivity [1]. ....

As discussed, the primary aim of this experiment is to apply and understand something of recombinant DNA technology. Students were therefore provided with both blue and white colony phenotypes that contained just one of the four possible fragments; in just one of its two orientations. The students’ task was to isolate the plasmid DNA and undertake the restriction digests and gel electrophoresis in order to resolve which fragment had been inserted. Fragment size is estimated by plotting a standard curve of known lengths of linearised DNA (kb) against their migration distance through the gel matrix (mm). .....

This study does not present any novel ideas. Rather, its role is to assist students in learning and applying generic skills in the field of molecular biology and genetics. ...

Experimental Procedures ...

show/hide

photoJill comments on the results (31s):

photoSarah talks about the challenges of writing the results and discussion (1m 18s):

Results

Fragment Size Determination: Student Gel

The student gel displayed bands of DNA from the blue colony phenotype (lanes 2-4), the white colony phenotype (lanes 7-10), and the HindIII reference standard (lanes 1, 6 and 11). The HindIII standard curve of 1/migration versus log10fragment size generated the linear equation: y = 58.379x – 0.246 (R2 = 0.99) (Figure 5). 

graph

Figure 5. Standard curve indicating log 10 fragment size against migration, constructed from measurements made from the student gel shown at Figure 6.

Band sizes were estimated using this equation. All digests of the blue colony phenotype yielded single bands of approximately 2.7 Kb. Two bands were visualized in the lane of undigested DNA. The EcoRI digest of white colony phenotype DNA yielded two bands (2.8 and 3.4 Kb). The BamHI digest of same yielded one band (5.8 Kb). Two bands were visualized ..... Through the gel, there are a number of ghost bands and bands of nicked/relaxed constructs. Refer to next section for description of these (Figure 6).  

bands

Figure 6: Student gel showing the each of the digested plasmid DNA samples and HindIII reference standards (lanes 1, 6, and 11). Lanes 2-5 contain DNA from the blue colony phenotype: double digest (DD), negative control (C), BamHI digestion (B), EcoRI digestion (E), in that order. Lanes 7-10 show some, but not all expected bands of DNA from ......

Fragment Size Determination: Model Gel

The faint (almost indistinguishable) banding patterns of the student gel were not entirely adequate for completing the required analyses. Therefore, a model gel will also be presented and discussed in this paper. .....

All digests of the blue colony phenotype yielded single bands of approximately 2.8 kbp. Several bands and smearing were visualized in the lane of undigested DNA. The EcoRI digest of white colony phenotype DNA yielded two bands (2.7 and 4.1 Kb). .... 

Spectrophotometric Analyses

In the student gel, the experimental concentration of DNA for each of the white and blue colony phenotype samples was 36.67 and 48.33 μg mL-1, respectively (4 sig. figs.). Each sample yield was therefore ~ 3.67 and 4.83 μg, again respectively (3 sig. figs.). The purity of DNA ...

show/hide

photoJill comments on the discussion (22s):

photoSarah describes how she could have improved her discussion (54s):

Discussion

Restriction mapping of all possible inserts (data not shown) reveals that the banding pattern of the model gel best resembles that of the 4878 Kb insert. Specifically, the predicted EcoR1 digest bands (4.9 and 3.0 Kb) are most consistent with the experimental fragment sizes (4.1 and 2.7 Kb). That these estimates are a little low is explained by the error inherent curve fitting, significant figures and manual measuring of the gel photograph. Gel fitting programs may assist in reducing some of this error.

The faint/indistinguishable banding for the white colony phenotype DNA used in the student gel is most likely the result of incorrect preparation of the sample. It could also be that the cells from which this DNA was taken ....

The 4.9 Kb λ-phage fragment has been inserted in orientation 2 (refer Figure 3). That is, ...

The blue colony phenotype DNA banding patterns were useful and as expected for both the student and model gel. They confirm that these samples do not have the insert present because of the absence of a second BamHI restriction site. ...

The productivity analyses (yield, concentration and purity) were not consistent with the manufacturer’s expected values (100 μg mL-1). This is most likely explained by ....

Negative control lanes contain undigested DNA. Hence the migration pattern is of supercoiled, slow moving constructs. These cannot be measured as linearised fragments. Smearing is the result of ... Ghost banding is caused by the presence of double-stranded, cyclic, colied DNA [5]. It is mitigated.... Nicked, open circular forms of DNA occur .....

This experiment has served as a platform for learning techniques in the application of recombinant DNA technology. It highlights some of the limitations of this technology (resolution, contamination, indirect observation of products) These are among many considerations that should be taken into account at every application of these techniques.

show/hide

photoJill suggests ways in which students can improve their report writing (36s):

References

[1] Hunt I (2005) From gene to protein: a review of new and enabling technologies for multi-parallel protein expression. Protein Expres. Purif. 40, 1-22.

[2] pBluescript®II Phagemid Vectors Instruction Manual (2003) Catalog #212205, #212206, #212207 and #212208 Revision #083001m. Copyright © 2003 by Stratagene.

[5] Hengen P N (1996) Eliminating ghost bands from plasmid preps. Trends Biochem. Sci., 21, 441-442.

» Screen 1