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. Moreover, the second transformation attempt resulted in an unsuccessful transformation due to inefficient uptake of DNA by the host organism, which might have been caused by several factors, such as improper preparation of competent cells, issues concerning the ligation reaction, or the transformation protocol itself.

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. Furthermore, troubleshooting in each area of competent cells preparation, ligation reaction and transformation protocol can assist in identifying and rectifying these challenges, such as re-evaluating the insert-to-vector ratio, incubation time, and temperature for the ligation reaction or testing and optimizing various transformation conditions.

Future experiments can investigate alternative restriction enzymes, plasmid vectors, and competent cell strains to improve the overall success of the genomic library construction process. Moreover, ensuring a suitable recovery time for transformed cells, providing a nutrient-rich medium during recovery, using appropriate plating methods and conditions, and including positive and negative controls in each transformation experiment will aid in enhancing the efficiency of the genomic library construction and subsequent functional selection of the streptomycin resistance-conferring rspL gene.
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. Moreover, the second transformation attempt resulted in an unsuccessful transformation due to inefficient uptake of DNA by the host organism, which might have been caused by several factors, such as improper preparation of competent cells, issues concerning the ligation reaction, or the transformation protocol itself.

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. Furthermore, troubleshooting in each area of competent cells preparation, ligation reaction and transformation protocol can assist in identifying and rectifying these challenges, such as re-evaluating the insert-to-vector ratio, incubation time, and temperature for the ligation reaction or testing and optimizing various transformation conditions.

For this experiment, the most likely problem would be with transformation. It could be optimized and modified by making changes mentioned below:
a. Temperature and duration: Confirm that the heat shock conditions (e.g. 42°C for 30-45 seconds) are appropriate for your cells.
b. Recovery duration: Allow adequate time (usually 1 hour) for the cells to recover and express the antibiotic resistance marker.
c. Optimal antibiotic concentration: Ensure the appropriate concentration of streptomycin for selection is used on the plates. Too high or low of a concentration may result in inadequate selection.

Future experiments can investigate alternative restriction enzymes, plasmid vectors, and competent cell strains to improve the overall success of the genomic library construction process. Moreover, ensuring a suitable recovery time for transformed cells, providing a nutrient-rich medium during recovery, using appropriate plating methods and conditions, and including positive and negative controls in each transformation experiment will aid in enhancing the efficiency of the genomic library construction and subsequent functional selection of the streptomycin resistance-conferring rspL gene.

don't change anything from the above information. just use the below mentioned references and do in-text citations:

Cirino, P. C., Mayer, K. M., & Umeno, D. (2003). Generating mutant libraries using error-prone PCR. Methods in molecular biology (Clifton, N.J.), 231, 3–9. https://doi.org/10.1385/1-59259-395-X:3
Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology, 166(4), 557–580. https://doi.org/10.1016/s0022-2836(83)80284-8
Inoue, H., Nojima, H., & Okayama, H. (1990). High efficiency transformation of Escherichia coli with plasmids. Gene, 96(1), 23–28. https://doi.org/10.1016/0378-1119(90)90336-p
Sorek, R., Lawrence, C. R., & Wiedenheft, B. (2013). CRISPR-Mediated Adaptive Immune Systems in Bacteria and Archaea. Annual Review of Biochemistry, 82(1), 237–266. https://doi.org/10.1146/annurev-biochem-072911-172315
Chen, I., & Dubnau, D. (2004). DNA uptake during bacterial transformation. Nature Reviews Microbiology, 2(3), 241–249. https://doi.org/10.1038/nrmicro844