Wet Lab Notebook
In this page, we offer a day-to-day analysis of our lab experiments, providing an in-depth look into the progression of our work. This section serves as a real-time documentation of our research journey, capturing the challenges encountered in the laboratory. In the beginning you will find a week to week summary of our work. In the “wet lab notebook”, you will find a full detailed description of our daily progress and actions in the lab, along with brief descriptions explaining both the results and the reason why we proceed to each experiment and alteration.
Laboratory Training
These weeks were dedicated to an intensive biosafety and laboratory training held in the Biology department of National Kapodistrian University of Athens by our instructor (Ms. Elena Pappa). The wet lab members got familiar with a variety of basic techniques and they learned how to design and execute a series of experiments.
Preparation of solutions and competent cells
Waiting for M13 Phage to arrive, necessary preparations were performed to save time for later. Solutions, mediums and chemically and electro-competent XL1-blue cells were made according to the appropriate protocols. Simultaneously, the majority of the experiments that were to be conducted, were carried out using PUC19 plasmid instead of the virus (e.g. digestion, ligation, chemical transformation, plasmid isolation, gel extraction). Thus, we were able to gain experience and optimize all of our protocols to the needs and behavior of XL1-blue cells.
Results:
M13 Manipulation, Chemical transformation
The M13KE vector arrived. It was diluted, digested and then loaded in electrophoresis gel in order to obtain the “reference image”. We proceeded to transformation of XL1-blue cells and then isolation of M13 from plaques. The first attempts of Mutagenesis PCR reactions were performed unfortunately with no expected results.
Results:
Mutagenesis PCR attempts and new primers
The PCR attempts continued and it was decided that new primers would be designed and ordered. However, we still did not get the results we wanted.
Results:
Mutagenesis and insertion PCR attempts and re-design
After various failed PCR attempts, we changed our strategy and agreed upon redesigning, using a different method to insert the desired mutations to M13KE.
We ordered 4 gBlock/Gene Fragments that would be inserted in the M13 phage with double digestion and ligation. While waiting for them to arrive, we prepared for our next steps by testing the efficiency of our enzymes, preparing competent cells, LB, petri dishes etc and reisolating our control plasmid (PUC19) while continuing to conduct PCR reactions. For these reactions, we used our new primers as well as digested M13KE using Pstl.
Results:
“Hippuric Acid” Gene Fragment Cloning
“Hippuric Acid” Gene Fragment arrived from Twist Biosciences and was resuspended. M13KE and Fragment's DNA was double digested (BsrGI & KpnI), ligated and used to transform chemically competent cells. Transformation was untrustworthy since negative control gave blue plaques and it was repeated twice being unsuccessful. In parallel the origin of the DNA of the first transformation was determined to be unmodified M13 DNA.
By the end of the week “Hippuric Acid 2” gBlock had arrived from IDT and it was resuspended.
Results:
“Hippuric Acid 2” gBlock Cloning - ”Eicosane”, “Octadecanal” and “Perilic Aldehyde” Gene Fragment Cloning - M13 amplification and purification.
“Hippuric Acid 2” gBlock had arrived from IDT. M13KE and gBlock’s DNA was double digested (HindIII & KpnI), ligated and used to transform chemically competent cells. The transformation was unsuccessful.
In the same week the Gene Fragments for Eicosane, Octadecanal and Perilic Aldehyde arrived from Twist Biosciences. M13KE and the Gene Fragments were double digested (BsrGI & KpnI), ligated and used to transform chemically competent cells. Again the transformation was unsuccessful.
The same week a vial of purified M13 bacteriophage was produced and its concentration was determined by titration. The purified bacteriophage was used to test tryptophan fluorescence in the presence and absence of hippuric acid.
Multiple repetitions of the transformations were made producing no result.
Results:
Transformation - “Hippuric Acid 2” gBlock Cloning - ”Eicosane”, “Octadecanal” and “Perillaldehyde” Gene Fragment Cloning - M13 amplification and purification - Silicon coated wafer production and test
To ensure that the plaques were indeed formed from modified phages, we isolated and digested with HindIII, DNA from several plaques.
At the same time, silicon gold-coated wafers were constructed and used to test the non phage-VOC affinity.