27/06/23 -> Two promotors were used (p_48 and p_IdhL1) to create the recombinant plasmid which was then ran on the Golden Gateway cloning program on the Thermocycler.

28/06/23 -> The recombinant plasmids were taken from the Thermocycler and a restriction digest was performed. We mixed the plasmid, Xbal and BCul enzymes, FastDigest green buffer and nuclease-free water*. The following program was used on the Thermocycler to cut the DNA using the enzymes stated.

1. 37°C for 15 minutes
2. 65°C for 20 minutes
3. 10°C hold

Reflections of this week

03/07/23 -> The recombinant plasmids were prepared again.
04/07/23 -> The restriction digest was carried out and the results were visualised under gel electrophoresis. We loaded 10µl of the plasmids instead of 5µl to ensure that there was enough DNA to be visualised. It was at this stage that we were told that the plasmid was not purified and that this was needed to have been completed before the restriction digest. We still looked at our gel and despite this, the image was clearer, and the recombinant plasmid was able to be visualised.

Reflections of this week

10/07/23 -> Competent DH5-α E. coli cells were prepared using this protocol.

12/07/23 -> The freeze-dried RIP DNA was made up to 12.5 ng/µl concentration then added this along with the other reagents to a PCR tube. Another tube was made using the same reagents along with the vector pX2800. Both were then added to the Thermocycler following the restriction digest protocol.

An agarose gel was prepared and once the Thermocycler program was completed, the vector was load and run down the gel. After the vector was purified by gel extraction. The weight of the gel was calculated to work out the volume of the binding buffer needed to make a 1:1 ratio. The gel was melted down and then purification using a GeneJet was carried out.

The next step was to the DNA insert ligation into the vector DNA. This was done using the T4 DNA ligase at 1 Weisse unit and adding the correct components to a PCR tube before loading onto the Thermocycler.

13/07/23 -> In preparation for transforming the ligated vector DNA into DH5-α competent cells, LB plates were poured. Once set the transformation was carried out, following this protocol.

24/07/23 -> For each of our 10 linear DNA, we cloned each with 3 different promoters: P32, P48 and Pldhoop. In addition a different CDS, a lacto terminator, buffer, ATP, T4 DNA ligase and BsaI was added to each. Also a RBS was added to those with P48 promotors. The reactions were left in the Thermocycler overnight.

25/07/23 -> The DNA was transformed into DH5-α E. coli and the petri dishes were left overnight.

26/07/23 -> The plates were checked on in the morning with all showing colonies apart from the 'his AHL acylase codon opt' with the P32 promotor.
Later, overnights were made of those successfully transformed.

27/07/23 -> A mini-prep of the overnights was performed following this protocol. An addition experiment ran parallel to this where mPapaya, mCherry, a positive control plasmid, a negative control and BFP were cloned.

07/08/23 -> The transformed L. plantarum plates were checked and to see where colonies had formed. From this, we made a plan for the this weeks next steps.
Overnights of the transformed L. plantarum were made using MRS laced with erythromycin and inoculating it with a single colony.

08/08/23 and 09/08/23 -> Western blots were carried out on the lysed cells of the transformed DNA following this protocol.
Also after speaking to our primary PI, we decided to clone some of the DNA sequences that failed to be transformed with a different plasmid backbone, pX_1645.

10/08/23 -> Unfortunately, the results of the western blot were unsuccessful with no bands showing. This left us thinked that either the L. plantarum wasn't expressing any of the proteins or something went wrong with the experiment.
Before doing the western blots, the OD₆₀₀ wasn't checked and so we were unaware to whether the bacteria was in the optimum growth phase. Our plan next week is to prepare overnights to check the OD is at the optimal and to then carry out another western blot.

The correct volumes needed for each component for redoing the failed transformations. Using these Golden Gateway assembly was used to clone the DNA and plated.

11/08/23 -> The transformation plates were verified for colony growth but as it was the end of the week, the mini-prep was held off until the following week and so the plates were left in the cold room over the weekend.

14/08/23 -> Overnights of both the successfully transformed L. plantarum and E. coli plates were made and stored in shaking incubators until the morning.

15/08/23 -> The OD of the overnights was calculated and the 24 hour cultures were prepared. These were left in the incubator in baffle flasks to promote the growth of L. plantarum through aeration.

The DNA was also mini prepped out of the DH5-α E. coli overnights. The concentrations were calculated for sequencing, however, these seemed lower than expected and we decided to redo the mini-prep using different concentrations.

16/08/23 -> The OD of the 24-hour cultures were recorded and the cultures were span down for the pellet to be resuspended and lysed. A western blot was carried out on the lysed cells, following this protocol.

The blot revealed bands in all samples, but it did not show that the L. plantarum was expressing our desired proteins due to there being a band present in the wild type. This meant that the Western blot was showing a non-specific binding of a protein normally found within L. plantarum with the antibodies.

The second mini-prep was carried out yet the results were no better than the previous day. Our PI wanted to mini prep herself to see if any mistakes had been made and so overnights were made for this. When making the overnights later, a slight contamination on the plates was noticed, the colonies were picked carefully. This may have been a reason for the low concentration values.

17/08/23 -> After the failure to detect the proteins yesterday, a western blot was conducted on the supernatant of the signal peptide DNA samples to see if the molecules had been exported out of the cell during the lengthy growth time. The blot was left on the iBind overnight.

Our PI did the mini-prep and managed to get a higher concentration and so the DNA could be sent off for sequencing.

18/08/23 -> The western blot of the supernatant was taken off the iBind and the steps were taken for visualisation of it. The membrane did not show any results with no bands on either the wild-type L. plantarum or the samples. To cover all avenues, a last western blot of the samples was undertaken using the whole cells from the stored pellet. This again did not yield any results, meaning we have exhausted all routes with these samples. Our next steps are to perform a western blot of the transformed C2925 E. coli to see if our DNA has been expressed in these E. Coli. It may be possible that the L. plantarum is not accepting the DNA and not expressing it, however, if it is not expressing in E. coli, it may mean that our transformations have not been successful.

21/08/23 -> Cloning was carried out with the promotors T7 and PJ23100 and all of the DNA was transformed into E. coli, DH5a. With the colonies, overnights were set up. E. coli (DH5a and C2925) was streaked in preparation for competent cell prep.
22/08/23 -> After transformation and overnights had been completed into DH5α, the OD₆₀₀ of the overnights were recorded and a whole cell western blot was conducted. The results were unclear due to the amount on the membrane, and in the bands that were visible, the bands did not correlate to the size of the desired proteins. Overall, this made the results inconclusive.
From the overnights, the DNA was mini-prepped out and used to make glycerol stocks.
23/08/23 -> Competent cells were made and the PJ23100 promoters with our DNA were re-transformed into DH5a again.
24/08/23 -> The C2925 DNA was re-transformed into C2925 to make up glycerol stocks.
25/08/23 -> The previous day's transformations had been successful and were left in the cold room over the weekend. Anything that was unsuccess in its initial tranformation into L. plantarum was transformed into E. coli then left in the static 30^oC incubator.