Tranfsorming Plasmids into E. coli and Culturing

Previously, Golden Gate and adding homologous arms to our plasmids (containing HA-GFP tags to visualise translation live) was conducted. Given the nature of science, not every plasmid would have taken up the homologous arms, therefore we need to isolate successful golden gate. To do this, the plasmids were transformed into E. coli (C2987H).

This process was straight forward, with a clear protocol all steps were conducted with ease. E. coli were thawed on ice, and a small amount of plasmid was added for each gene. this mixture was left to incubate on ice before being heat shocked at 42C in a water bath. This was to ensure maximum amount of plasmids were taken up by the bacteria. A small volume of LB was added to the mixture and left to incubate for another hour whilst being shaken. Finally, bacteria was then spread onto LB plates, containing chloramphenicol - an important antibiotic as bacteria without the plasmid would die, however those with the plasmid would have chloramphenicol resistance, allowing me to select for bacteria with the plasmid. These plates were grown overnight at 37C.

The next day I was expecting to see some single colonies however for a long time, doing several repeats of this method, very few/no colonies were observed! This was quite frustrating as each repeat takes a long time, therefore it felt like a lot of time was being wasted by repeated. My post-doc and I decieded to change small details with each repeat to see what may be causing a low number of colonies, with changes including different annealing buffer and ligase buffers. Ultimately, the experiment worked by diluting the first annealing step to ensure when I got to the golden gate stage, the correct volume of homologous arms were added to the mix.

Few single colonies on APX-1 plate (test)

Despite there not being as many colonies as desired, some colonies were better than none, and we decided to proceed with the project!

Now that I had single colonies of bacteria with the plasmid containing the desired insert, the next step is to culture these bacteria to increase the amount of plasmids that we have. This was done by making bacteria cultures.

For each gene, 6 cultures were made, making an overall 18 cultures as I was working on 3 genes. To make the cultures, 2.5ml of LB was added to a tube as well as a small volume of chloramphenicol. For each culture, I used a 200 ul tip and tapped a single colony and dropped the tip into a tube contianing LB. This step was a little difficult given the single colonies were quite small and only a single tap was required, allowing me again to practice my fine motor skills. As well as this, this process was done 18 times so it waas challenging keeping my hand steady for all 18 cultures!

Once all cultures were made, these tubes were incubated overnight at 37C at 250 rpm (16 hours) to allow the bacteria to grow.