Discussion:

The primary objective of this project was to create a genomic library using a streptomycin-resistant strain of E.coli Top10 (4537 bp) as the genomic DNA, incorporating it into a pUC19 plasmid (2686 bp) vector. The successful construction of this genomic library was expected to confer streptomycin resistance to a recipient E.coli strain Mach1 through the functional selection of the rspL gene. However, throughout the experiment, several challenges were encountered, leading to changes in the experimental design and troubleshooting of errors.

An initial gel electrophoresis performed to confirm the presence of the rspL gene after digestion of genomic DNA with BamHI resulted in no visible bands. This was primarily due to the low concentration of the originally isolated genomic Top10 DNA (33.2 ng/µL), which was further diluted during the digestion reaction and loading of the gel. This concentration was below the minimum detection limit required for the INtRON RedSafeTM nucleic acid stain to work efficiently (50 ng/µL). As a consequence, no bands were detected, and the experiment had to be repeated with a new and cleaned up genomic DNA sample.

Subsequent gel electrophoresis performed after digesting the cleaned-up genomic DNA with BamHI showed successful digestion of the genomic Top10 DNA, as evidenced by a shorter smear in comparison to the uncut band of the genomic DNA. In addition, the plasmid pUC19 showed two very faint bands after digestion with BamHI, indicating appropriate digestion of the plasmid as well.

A ligation reaction was set up using a 1:1 ratio for cut genomic Top10 DNA (insert) and cut plasmid pUC19 (vector). After overnight incubation at 4 °C, the ligated product was transformed into Mach1 chemically competent cells. The initial transformation results were inconclusive due to contamination observed in the positive control plate. A second transformation attempt also resulted in inefficient uptake of DNA by the host organism, leading to an unsuccessful transformation.

To increase the likelihood of obtaining a fragment containing the rspL gene, a third gel electrophoresis was performed using HindIII in addition to BamHI for digesting both genomic Top10 DNA and plasmid pUC19. The use of two different enzymes would increase the accuracy of the construct and facilitate the incorporation of the desired fragment into the plasmid.

Based on the information provided by the restriction analyzer, the BamHI enzyme cuts at two different sites on genomic DNA (180 and 2865), producing large fragments that appeared as a smear in the gel electrophoresis images. HindIII was introduced to cut at an additional site (4130) on genomic DNA, increasing the likelihood that the fragment containing the rspL gene would be successfully incorporated into the plasmid.

In summary, the construction of a genomic library using E.coli Top10 genomic DNA and pUC19 plasmid DNA encountered several challenges throughout the experiment, including low DNA concentrations, contamination, and inefficient transformation. The introduction of the HindIII enzyme in addition to BamHI for restriction digestion was expected to increase the probability of obtaining a fragment containing the rspL gene. Further optimization of ligation and transformation protocols may be necessary to enhance the efficiency of the genomic library construction and subsequent functional selection of the streptomycin resistance-conferring rspL gene. Future experiments can also investigate alternative restriction enzymes, plasmid vectors, and competent cell strains to improve the overall success of the genomic library construction process.



I do not want step by step solution. Take the information from below and add it into the discussion itself mentioned above and do not use numbering format.

The second transformation attempt resulted in an unsuccessful transformation due to inefficient uptake of DNA by the host organism. This might have been caused by several factors, such as improper preparation of competent cells, issues concerning the ligation reaction, or the transformation protocol itself. Troubleshooting in each of these areas can be implemented to identify and rectify these challenges further:

1. Quality and efficiency of competent cells: Ensure that the competent cells used are in good condition, have high transformation efficiency, and have been stored properly. If required, prepare a new batch of competent cells or consider using commercially prepared competent cells with high transformation efficiency. Additionally, test the competency of the cells with a known plasmid to ensure that they are suitable for transformation.

2. Ligation reaction optimization: Re-evaluate the insert-to-vector ratio, incubation time, and temperature for the ligation reaction. The success of a ligation reaction depends on an optimal molar ratio of insert and vector DNA as well as the concentration and activity of the T4 ligase enzyme. Optimization of ligation conditions can help ensure that the recombinant plasmid is formed effectively.

3. Transformation protocol optimization: The transformation efficiency is highly dependent on the methods employed, such as the temperature and duration of the heat shock. Therefore, it might be necessary to test and optimize various transformation conditions, including different heat shock times or temperatures, to obtain the most efficient transformation.

4. Recovery period and medium: Ensuring a suitable recovery time for transformed cells before plating is essential, as it enables the cells to repair and express antibiotic resistance markers. Moreover, it is crucial to provide a nutrient-rich medium during recovery to promote cell growth and increase the chances of successful transformation.

5. Plating methods and conditions: Ensure that the transformed cells are plated on appropriate selective media with accurate antibiotic concentrations. This will ensure selective pressure for the host organism to uptake the ligation product containing the rspL gene. Additionally, ensuring that the transformation plates do not contain any contamination and have been spread evenly and allowed to solidify is important.

6. Use of positive and negative controls: Including positive and negative controls in each transformation experiment can help identify potential issues with the experiment, such as contamination or problems with the competent cells or recovery conditions.