We visited the Enteric Disease Division at KMC, Manipal and observed the protocols for culturing, storage and detection of Vibrio parahaemolyticus strains under supervision.

• Primary Sub-culturing of E. coli DH5α.
• Quantifying growth of Primary culture of DH5α

Result
OD₆₀₀ = 2.5

• Gram staining was performed for the first subculture.

Result
E. coli DH5α cells were observed to be pink.
Cell debris were observed.

Troubleshoot
Initial smearing could be done more gently.

• Secondary subculture was performed
• Quantifying growth of secondary subculture of the culture

Result
OD₆₀₀ = 1.4

• Gram staining was performed for the secondary subculture

Result
E. coli DH5α cells were observed to be pink.
Some crystal violet stain residues were observed
Many cells were clumped together
Lesser cell debris were observed

Troubleshoot
Washing with water can be done better and longer.

• Multiple subcultures were prepared, and E. Coli DH5α growth was confirmed using Indole test and Gram staining

• Primary culturing of pET-22b(+) transformed E. coli DH5α from prepared glycerol stock.
• Quantification of transformed E. coli culture.

Result
OD₆₀₀ = 3

• Secondary culturing of E. coli DH5α.
• Quantification of transformed E. coli

Result
OD₆₀₀ = 0.7

• Plasmid isolation of pET-22b(+) from transformed E. coli (Trial 1)
• Quantification of Plasmid DNA.

Result
DNA concentration was found to be 38.1 ng/μL.

• Gel electrophoresis to confirm the presence of isolated plasmid.

Result
- The plasmid was not observed.
- No RNA contamination was observed.

Troubleshoot
- The culture was advised to be kept until an OD₆₀₀value of 1-1.2 was obtained.
- The concentration of plasmid should be around 100 ng/μL.

• Primary subculturing of pET-22b(+) transformed E. coli DH5α from prepared transformed plates.
• Quantification of transformed E. coli culture

Result
DH5α
-> Culture 1 OD₆₀₀ = 0.4
-> Culture 2 OD₆₀₀ = 0.6

• Secondary overnight sub-culturing of pET-22b(+) transformed E. coli
• Plasmid isolation of pET-22b(+) from transformed E. coli (Trial 2)
• Quantification of Isolated plasmid.

Result
DNA concentration was found to be 69 ng/μL.

• Gel electrophoresis for Isolated Plasmid

Result
The plasmid was successfully isolated.

• Primary Subculturing of pET-22b(+) transformed E. coli DH5α
• Quantification of Transformed E. coli

Result
Glycerol stock culture OD₆₀₀ = 2.1
Plate culture OD₆₀₀not measurable

• Plasmid isolation and checking sample concentrations
• Checking plasmid DNA concentration using nanocuvette

Result
The concentration of DNA isolated from plate culture was found to be 48.2 ng/μL.
The concentration of DNA isolated from stock culture was found to be 18.3 ng/μL.

• Gel electrophoresis for isolated plasmid samples

Result
The plasmid was successfully isolated.

• Single digestion of pET-22b(+). Was performed to check the efficiency of our enzymes. Reaction 1 was performed using NdeI enzyme. Reaction 2 was performed using the XhoI enzyme.
• Gel electrophoresis of single Digested pET-22b(+)

Result
The digested band was in the 5.5kb range according to the DNA ladder, while the plasmid itself is 5493bp. Thus, single digestion of pET-22b(+) using NdeI and XhoI was successful.

• Double digestion of pET-22b(+)
• Gel electrophoresis of double digested pET-22b(+) samples
• Primary Subculturing of pET-22b(+) transformed E. coli DH5α
• Quantification of Transformed E. coli

Result
- Plate culture 1: OD₆₀₀ = 0.2
- Plate Culture 2: OD₆₀₀ = 0.6

• Plasmid Isolation of pET-22b(+) from E. coli DH5α
• Concentrations of Isolated plasmid

Result
The concentration of DNA from plate culture 1 was found to be 54.4 ng/μL.
The concentration of DNA from plate culture 2 was found to be 48.8 ng/μL.

• Gel electrophoresis of isolated plasmid samples
• Dissolving lyophilized IDT MAM7 gene fragment

Result
The concentration of isolated plasmid was found to be 11.3 ng/μL.

• PCR Amplification of MAM7
• Running of gel electrophoresis of amplified MAM7
• Purification of MAM7 amplicon using PCR cleanup kit

Result
After purification, the concentration of purified MAM7 was way too low.

• pET-22b(+) Plasmid Isolation using Miniprep Kit

Result
Plasmid DNA concentration from plate culture 1 was found to be 55.1 ng/μL.
Plasmid DNA concentration from plate culture 2 was found to be 56.9 ng/μL.

• PCR Amplification of the MAM7 gene
• Dissolving lyophilized IDT MAM7 gene fragment

Result
OD₆₀₀ = 1.3 ng/μL.

• MAM7 Primer Synthesis for AMplification of gene fragment
• PCR Reaction Mix Prep for MAM7 gene amplification
• PCR Gel Purification of Amplified MAM7
• pET-22b(+) and MAM7 Ligation
• Chemical Preparation for PEG Method
• Competent cell preparation using the PEG method

Result
The molar concentration of glucose and Mg²⁺ was rectified for the TSS preparation needed for competent cell preparation and transformation.

• Transformation of pET-22b(+) was performed via PEG Method (Trial 1)

Result
We determined the transformation efficiency and we obtained the highest number of isolated colonies. Hence, it was successful.

• Visualizing MAM7 ligated pET-22b(+)
• Transformation of MAM7 ligated pET-22b(+) to E. coli DH5α by PEG method (Trial 2)

Troubleshoot
Since the competent cells we prepared were a day old despite being stored at -80°C. Hence we assumed this to be the reason for our failed transformation

• Competent Cell preparation using the CaCl₂ method (Trial 1)
• Transformation of MAM7 ligated pET-22b(+) to E. coli DH5α by CaCl₂ method (Trial 1)
• Competent cell preparation (Trial 2)
• Colony PCR (Trial 1 of CaCl₂ method) = 5 mixtures from 1:3

Result
-The vector to insert ratios yielded proper
- Colonies 1,2,4,6 and 10 showed the best
- The MAM7 amplicon band (At around 2.5kb) was distinct for these colonies and showed the least number of non-specific bands.

• Running of gel for Colony PCR (Trial 1)

Result
No insert was observed. Instead, a big smear was observed at that location.

• Transformed pET-22b(+) plasmid Isolation using Miniprep Kit

Result
During the gel running, two bands were observed corresponding to one circular and one linear plasmid. But on checking for the concentration, the value was too low.

• Colony PCR (Trial 2 of CaCl₂ method) = 3 mixtures from 1:2, 4 reaction mixtures from 1:3 transformed plates.
  -> 3 reaction mixtures from 1:2 transformed plates
  -> 4 reaction mixtures from 1:3 transformed plates

Result
After running the gel, we observed 3 bands, out of which we observed one band exactly below the insert. The reason for the other two nonspecific bands observed is yet to be troubleshot.

• Transformed pET-22b(+) Isolation using Miniprep Kit

Result
We obtained a good concentration of the plasmid. After running the gel, we could observe only one single linear band.

• Single digestion of the transformed pET-22b(+) ligated with MAM7 using NdeI was carried out.
• We ran the gel and confirmed that our enzymes were working.
• Double Digested transformed pET-22b(+) ligated with MAM7 using NdeI and XhoI Enzymes
• Gel extraction was performed

Result
- 3/4th of the mixture was subjected to gel electrophoresis.
- After gel extraction, we obtained the concentration of pET-22b(+) and MAM7 after double digestion i.e. 5.5ng/μL and 5.6ng/μL, respectively.

• PCR Purification

Result
- We obtained higher concentrations via PCR purification than Gel extraction.
- pET-22b(+) =17.1ng/μL, 17.7ng/μL=MAM7; were the obtained concentrations.

• pET-22b(+) and MAM7 Ligation
  - Higher ratios for vectors to insert were considered
  - Vector to Insert ratios that were ligated = 1:4(Gel Extracted), 1:5, 1:6(PCR Purified)
• Competent Cell preparation using the CaCl₂ method (Trial 2)
• Transformation of MAM7 ligated pET-22b(+) to E. coli DH5α by CaCl₂ method (Trial 2)