Background information
This portion of the lab section involved creating a genomic library using a streptomycin resistant strain of E.coli top10 (4537 bp)  as the genomic DNA which will be  incorporated into puc19 plasmid (2686 bp) vector as proposed earlier in PART B proposal document. To achieve this our pair (Jetul and Japneet) firstly isolated the DNA from genomic stain and plasmid using specific isolation kits included in method section. Followed by restriction enzyme digestion with suitable enzyme prior to ligation. The plan then included functional selection based on streptomycin resistance which will confirm successful insertion of rspL gene in the recipient E.coli strain Mach1, the competent cells used for transformation.
To achieve the proposed plan, E.coli strain top10 (4537 bp) served as the genomic strain that will be the source of streptomycin resistance rspL gene. The FastDigest HindIII (thermo scientific) digested fragment will be insert into a digested pUC19 plasmid vector (2686 bp) utilizing ligation process. Once ligated, the product will be transformed with Mach1 chemically competent cells (O.D. value = 0.77). To confirm the success of ligation the transformed product will be plated on ampillicin/X-gal/IPTG plates and the white colonies will then be streaked onto streptomycin plates to verify the rspl gene streptomycin resistance property. 
As per the proposal, digesting the bacterial strains with similar digestion enzyme (HindIII) prior to ligation followed by transformation would have ideally given the genomic library however, throughout the project certain decisions were changed based on the results which will be thoroughly covered in this document along with the solutions taken to troubleshoot the mistakes.

Methods/Materials
Isolation of genomic DNA and plasmid DNA:
To start with the procedure of creating a genomic library from E.coli strain Top10 and plasmid pUC19, 5 mL genomic DNA (Top10) and 3 mL plasmid pUC19 was isolated using GeneJET genomic DNA purification kit (lot 01319594) and GeneJET plasmid Mini prep kit (lot 01218820)  respectively. This task was completed in pair, genomic DNA isolated by Jetul and pUC19 by Japneet. 
Concentration of both genomic and plasmid pUC19 were determined using SimpliNano which are summarized in the table below.
Table 1: Observed concentrations of isolated genomic material using SimpliNano.
Genomic material	A260/A280	A260/A230	DNA Conc. (ng/µL)
Top10	1.988	1.644	33.2
pUC19	1.866	1.211	17.2
The above table summarizes the observed concentration data for isolated genomic material on Feb 21, 2023. The volume of sample loaded in SimpliNano was 2 µL for both DNA. Elution buffer(01177350 and 00991597) from both respective isolation kits were used as a blank on SimpliNano.  
Restriction Enzyme Digestion:
As per part A proposal our group wanted to use HindIII enzyme of restriction enzyme digestion, but BamHI was used instead. This was changed because the aim was to check whether BamHI will show the same digestion or similar results with strainTop10 of E. coli as it was the case with K12 strain.
Both genomic DNA from Top10 and plasmid DNA of pUC19 were digested with BamHI enzyme in second week of this project (March 8, 2023) and the reagent volumes used are listed in the table given below.
Table 2: Reagents for restriction enzyme digestion of genomic material.
Digestion Reagents	pUC19	Top10 (rspL gene)
Genetic material	2 µL	10 µL
Fast digest buffer	2 µL	2 µL
BamHI (Fast Digest Enzyme)	1 µL	1 µL
PCR Grade Water	30 µL	22 µL
Total Reaction volume 	35 µL	35 µL
This table summarizes the reagent recipe used for first restriction enzyme digestion of genetic materials used to create the genomic library. The above listed both digestion reactions were incubated in 37 °C water bath for 30 minutes before heat inactivation at 80 °C for 10 minutes.
 gel electrophoresis
 This gel electrophoresis was performed on same day in order to confirm whether the digestion was successful or not and whether the isolated genomic DNA contains the rspL gene. To prepare this, 1% gel was prepared using 500 mg Agarose powder in 50 mL 1X TAE buffer with 2.5 µL INtRON RedSafeTM for visualization.
1Kb DNA ladder was used as a standard with 6X loading dye. 10 µL of digested genomic was loaded into the gel. Gel was run for 20 minutes at 120V. When analyzed under UV light, there were no bands visible.
DNA clean-up:
Since gel electrophoresis results indicated no bands it was decided with the help of lab instructor to perform a DNA clean up for genomic to concentrate it more.
Originally isolated genomic Top10 DNA was cleaned up using “Thermo Scientific GeneJET Gel Extraction and DNA clean up kit” the following week ( March 14, 2023). A small change was introduced in original clean up protocol which stated “10 µL of Elution buffer” to elute which was updated to “15 µL of Elution Buffer”. Concentration of genomic DNA was checked on SimpliNano after clean up which came out to be significantly low ( 0.012 µg/ µL) as compared to earlier 33.2 ng/ µL. This indicated the DNA was lost in clean up process.
Table 3: Concentration of genomic DNA (Top10) analyzed on SimpliNano after DNA clean up.
	Cleaned up genomic Top10 DNA
A260/A230	0.826
A260/A280	1.609
ng/ µL	12
The above table lists the concentration of originally isolated genomic DNA of strain Top10 after clean-up using Thermo Scientific GeneJET Gel Extraction and DNA clean up kit (LOT 2599306). Volume of sample loaded on SimpliNano was 2 µL and the blank used was the elution buffer(LOT 01307087) from the DNA clean up kit.
New isolated genomic Top10 DNA provided by Vanessa:
Since the whole genomic DNA was lost therefore another vial of isolated genomic was provided by lab instructor. The concentration of new genomic DNA was analyzed on SimpliNano which came out to be 28.1 ng/ µL with 1.598 (A260/A280) and 1.143 (A260/A230). This genomic DNA was then cleaned-up using the same clean up kit with same modification of 15  µL elution buffer. After clean up the concentration was checked on SimpliNano and is stated in table below.



Table 4: Concentration of new genomic Top10 DNA before and after clean-up.
	Before clean up	After clean up
A260/A280	1.598	1.794
A260/A230	1.143	2.188
ng/ µL	28.1	109.6
The above table summarizes the observed concentrations of new isolated genomic top10 DNA provided by Vanessa. These concentrations refer to the DNA before using GeneJET genomic DNA purification kit with its corresponding elution buffer as blank. Also after clean up using Thermo Scientific GeneJET Gel Extraction and DNA clean up kit (LOT 2599306). Volume of sample loaded on SimpliNano was 2 µL and the blank used was the elution buffer(LOT 01307087) from the DNA clean up kit.
New Digestion reaction set up with cleaned up genomic DNA:
The new digestion was performed using the cleaned up genomic DNA with higher concentration. The table summarizes the reaction reagents and volumes.
Table 5: The reaction reagents for restriction enzyme digestion of both genomic material.
	pUC19	Top10 genomic DNA
Concentration	17.2 ng/ µL	109.6 ng/ µL
Genomic material	4 µL	5 µL
Fast Digest Buffer	2 µL	2 µL
Fast Digest Enzyme (BamHI)	1 µL	1 µL
PCR Grade water	28 µL	27 µL
Total reaction volume	35 µL	35 µL
The table gives the reaction volumes that were used to perform enzyme digestion of both genetic materials used in this project to construct a genomic library. Both reactions were incubated for half an hour in 37 °C water bath prior to heat inactivation at 80 °C for 10 minutes. The digestion reactions were then stored in ice bucket until gel electrophoresis apparatus was ready.
Another 1% gel electrophoresis:
Similar recipe of 1% gel electrophoresis was prepared that is 500 mg Agarose in 50 mL 1X TAE buffer along with 2.5 µL INtRON RedSafeTM for visualization. Same 1Kb DNA ladder was used as standard with 6X loading dye. This time since the concentration of newly cleaned genomic DNA was significantly high, only 5 µL of cut and uncut genomic along with cut plasmid with 6X loading dye was loaded. 
6X loading dye sample calculation:
1/(6 )=  x/(x+5)
x+5= 6x 
                                                                          
5= 6x-x 
5= 5x 
x=1 µL , where x is the amount of loading dye added to the sample.
The 1% gel was shared with another group with ladder in the center followed by uncut genomic, cut genomic and cut plasmid from our samples. First half of the gel was utilized by Mahan’s group. The gel was run for about 20-25 minutes at 120 V and the results were first visualized under UV light and then under BIOrad software which will be added in result section.
Ligation with only one reaction:
This was decided to check if the ligation was successful or not which was performed the following week (March 21, 2023). To do so, a 1:1 ratio of cut plasmid pUC19 and cut genomic top10 (insert) was ligated using T4 ligase (vector) accompanied with the use of PEG4000. 
The following table summarizes the reaction reagents and volumes.
Table 6: Ligation reaction set up for digested insert and vector using T4 ligase.
Reaction reagents	Ligation ratio (1:1)
Insert (digested genomic top10 DNA)	3 µL
Vector (digested pUC19)	3 µL
T4 Ligase Buffer (5X)	5 µL
T4 Ligase	2 µL
PEG4000	2 µL
PCR Grade water	10 µL
Total reaction volume	25 µL
This ligation reagent recipe was used to ligate the insert and vector achieved from restriction enzyme digestion of genomic top10 and pUC19 with BamHI. This ligation reaction was incubated overnight at 4 °C and heat inactivated after 24 hours at 65 °C for 10 minutes. The ligated product was then stored at -20 °C until transformation.
Transformation with Mach 01 strain competent cells:
Transformation of the ligated product was performed the same week (March 24, 2023). To proceed with transformation, 4 vials of competent cells (50 µL each) were provided by Vanessa along with 4 agar plates.
Goal was to plate one positive control, one negative control for validation of results with one experimental in duplicates. Protocol of transformation was used to first thaw the competent cells on ice (about 20 minutes) followed by setting up controls and experimental.
Table 7 : Controls and experimental reaction set for transformation of ligated product.
Reaction	Positive control	Negative control	Experimental
Reagents	50 µL competent cells + 10 µL pUC19	50 µL of competent cells 	50 

The transformation reactions were then incubated on ice for 30 minutes prior to heat shock at 42 °C for 30 seconds. The reactions were then placed on shaker for 1 hour until recovered. Meanwhile, when 30 minutes were left while cells were recovering, 4 agar plates were spread with 100 µL of 25 mg/mL Ampicillin, 40 µL of 20 mg/mL X-gal and 8 µL of 500 mM IPTG per plate respectively. After successive completion of 1 hour recovery of cells, one little change was introduced in which the cells were pelleted at 5000 rpm for 5 minutes. Instead of plating 200 µL onto plates, 150 µL was plated for each. This was done because in part A the positive control did not show TNTC which means.
Once solidified, all 4 plates were incubated at 35 ° C for 24 hours. Plates were retrieved from incubator the next day (March 25, 2023). 














Results: 

First gel electrophoresis.

Image 1: Picture of first 1% gel electrophoresis performed to confirm the presence of rspL gene after digestion of genomic DNA with BamHI













The above image was captured from UV light analysis of the 1% agarose gel prepared in 1X TAE buffer with 2.5 µL INtRON RedSafeTM and 6X loading dye. The well labelled 1 was utilized for 1Kb plus DNA ladder RTU (FroggoBI0- BIOHELIX), 5 µL loaded as standard whilst the well labelled 2 contains the digested genomic DNA (10 µL digested sample  + 2 µL loading dye). The above gel electrophoresis was run at 120 V for 20 minutes. Genomic uncut was not loaded which was considered an error. Moreover, there were no bands at all and the problem was low concentration of genomic DNA.

It was noticed that since INtRON RedSafeTM  requires at minimum 50 ng/ µL of the sample concentration to give any visualization detection effects, the above gel electrophoresis was unsuccessful. This is because the concentration of originally isolated genomic Top10 DNA was already quite low with 33.2 ng/ µL and while preparing the digestion reaction with total volume of 35 µL we used 10 µL of the genomic DNA which implies that our genomic was diluted. Not only this when we loaded 10 µL digested sample with 2 µL loading dye it further diluted. As per this, the concentration of loaded sample was 33.2 ng/ µL which is very less than 50 ng/ µL as per the RedSafeTM to work efficiently.
Calculations of digested sample concentration:
(33.2 ng / µL × 10 µL in digestion) / (10 µL while loading) = 33.2 ng/ µL
Hence, nothing was detected.
Furthermore, digested pUC19 plasmid and uncut genomic Top10 was not loaded and therefore nothing was there to compare which was a mistake.

This resulted in cleaning up the genomic DNA to get better concentration numbers and then perform digestion and gel electrophoresis for confirming the presence of rspL gene and action of BamHI. Another gel electrophoresis was performed with new genomic DNA provided by Vanessa followed by its clean up since the originally isolated genomic DNA was lost in clean up procedures.















Image 2: Second 1% gel electrophoresis performed after digesting newly cleaned genomic DNA.






 










The above image represents the picture captured of 1% gel electrophoresis run for 25 minutes at 120 V under UV light. This is the seconds gel that contained new digestion reaction containing the genomic DNA after clean up. The gel was prepared in 1X TAE buffer with 2.5 µL INtRON RedSafeTM along with 6X loading dye used for sample preparations. Gel was shared with another group (Mahan’s) which is represented with the arrow head and samples in lane 1,2,3 belongs to other group. Well labelled 4 contained the 5 µL of 1Kb plus DNA ladder RTU (FroggoBI0- BIOHELIX) ,  well 5 contained the uncut genomic Top10 (5 µL + 6X loading dye), well 6 contained cut genomic with BamHI((5 µL + 6X loading dye) and well 7 contains the cut plasmid with BamHI ((5 µL + 6X loading dye).



Image 3: BIORAD image of 1% gel electrophoresis performed for confirming the action of BamHI on rspL gene of genomic DNA and on plasmid pUC19.
















The above image represents the picture captured of 1% gel electrophoresis run for 25 minutes at 120 V under BIORAD Imager. The gel was prepared in 1X TAE buffer with 2.5 µL INtRON RedSafeTM along with 6X loading dye used for sample preparations. Gel was shared with another group (Mahan’s) which is represented with the arrow head and samples in lane 1,2,3 belongs to other group. Well labelled 4 contained the 5 µL of 1Kb plus DNA ladder RTU (FroggoBI0- BIOHELIX) ,  well 5 contained the uncut genomic Top10 (5 µL + 6X loading dye) which as expected showed a large uncut band, well 6 contained cut genomic with BamHI((5 µL + 6X loading dye) which showed a large smear in image and is shorter as compared to the large band of genomic in lane 5 hence suggest the digestion to be complete and well 7 contains the cut plasmid with BamHI ((5 µL + 6X loading dye) which showed two very faint bands as highlighted with red color on image.



Image 4: Transformation results containing experiment results and controls after 24-hour incubation at 37 degrees Celsius. 

The above image represents the picture captured of agar plates after 24-hour incubation at 37 degrees Celsius. Each of the plates above contains 100 µL of 25 mg/mL Ampicillin, 40 µL of 20 mg/mL X-gal, and 8 µL of 500 mM IPTG. Negative control contains 50 µL of competent cells. Positive control contains 50 µL of competent cells and 5 µL of pUC19. Experiment plates are in duplicate and contains 50 µL of competent cells and 10 µL of ligated product. In the experiment cells were heat shocked for 30 seconds at 42 degrees Celsius. 500 µL of broth was added to cells as medium. Cells recovered at 37 degrees Celsius for 1 hour. After recovery, cells were spun down for 5 minutes at 5000 x g and 200 µL of it was thrown. 150 µL of cells were spread onto the plates using Pasteur pipette. Bunsen burner was used to maintain a sterile environment. The experiment failed since the positive control contains contamination (the big white colonies shown in the image are contamination). 

Image 5: Transformation results containing experiment results and controls after 24-hour incubation at 37 degrees Celsius. Experiment was performed due to failure of the first one.
 
The above image represents the picture captured of agar plates after 24-hour incubation at 37 degrees Celsius.  Each of the plates above contains 100 µL of 25 mg/mL Ampicillin, 40 µL of 20 mg/mL X-gal, and 8 µL of 500 mM IPTG. Negative control contains 50 µL of competent cells. Positive control contains 50 µL of competent cells and 5 µL of pUC19. Experiment plates are in duplicate and contains 50 µL of competent cells and 10 µL of ligated product. In the experiment cells were heat shocked for 30 seconds at 42 degrees Celsius. 500 µL of broth was added to cells as medium. Cells recovered at 37 degrees Celsius for 1 hour. After recovery, cells were spun down for 5 minutes at 5000 x g and 200 µL of it was thrown. 150 µL of cells were spread onto the plates using Pasteur pipette. Bunsen burner was used to maintain a sterile environment. The experiment was not successful due to plating issue. There is inefficient uptake of DNA by host organism which led to inefficient transformation that mean unsuccessful transformation.

Image 6: Third 1% gel electrophoresis performed using HindIII and BamHI.

















The above image represents the picture captured of 1% gel electrophoresis run for around 45 minutes at 120 V under UV light. This is the third gel that contained a new digestion reaction containing the genomic DNA. The gel was prepared in 1X TAE buffer with 2.5 µL INtRON RedSafeTM along with 6X loading dye used for sample preparations. Well labelled 1 contained the 10 µL of 1Kb plus DNA ladder RTU (FroggoBI0- BIOHELIX), well labelled 2 contained the undigested genomic Top10 (10 µL + 6X loading dye), well labelled 3 contained digested genomic with BamHI and HindIII (10 µL + 6X loading dye), well labelled 4 contains the undigested plasmid (10 µL + 6X loading dye) and well labelled 5 contains the digested plasmid with BamHI and HindIII (10 µL + 6X loading dye).

Image 7: Information regarding the total length of the genomic Top 10 DNA using restriction analyzer
 
The above image provides information related to the total length of the genomic Top 10 DNA that is 4537 bp. The tool used for this was restriction analyzer. It also shows that BamHI cuts at 2 different sites on genomic DNA. The cutting positions are 180 and 2865. These sites give very big fragments that can be seen as smear in image 2. 
This enzyme cuts at 662 on pUC19 and creates sticky ends. The total size of the plasmid is 2686 bp. If we incorporate this plasmid inside a host mentioned above, it gives a smear. There are also chances that host could not uptake the DNA containing rspL gene.

Image 8: Information regarding the total length of the genomic Top 10 DNA and cutting positions using HindIII and BamHI enzymes using restriction analyzer.
 
The above image provides information related to the total length of the genomic Top 10 DNA that is 4537 bp. The tool used for this was restriction analyzer. It also shows that BamHI cuts at 2 different sites on genomic DNA and HindIII cuts at 4130 site on genomic DNA. The cutting positions for BamHI are 180 and 2865 and for HindIII is 4130. We used HindIII to make sure that our fragment contains rspL gene.

Image 9: Information regarding the total length of the plasmid DNA using restriction analyzer
 
The above image provides information related to the pUC19 plasmid DNA that is 2686 bp long. The tool used for this was restriction analyzer. It also shows that BamHI cuts at 662 position on plasmid DNA and HindIII cuts at 632 positions. Since we are using two different enzymes, there is a better chance that our fragment contains 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 (Cirino et al., 2003). 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 (Hanahan, 1983). 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 (Inoue et al., 1990). 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 (Sorek et al., 2013). 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) (Chen & Dubnau, 2004). 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 (Inoue et al., 1990).

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 (Cirino et al., 2003). The use of two different enzymes would increase the accuracy of the construct and facilitate the incorporation of the desired fragment into the plasmid (Hanahan, 1983).

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 (Sorek et al., 2013). 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 (Chen & Dubnau, 2004).

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 (Inoue et al., 1990). 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 (Cirino et al., 2003). 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 (Hanahan, 1983).

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 (Inoue et al., 1990).
b. Recovery duration: Allow adequate time (usually 1 hour) for the cells to recover and express the antibiotic resistance marker (Sorek et al., 2013).
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 (Chen & Dubnau, 2004).

Future experiments can investigate alternative restriction enzymes, plasmid vectors, and competent cell strains to improve the overall success of the genomic library construction process (Cirino et al., 2003). 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 (Hanahan, 1983).

Conclusion
 
References
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



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