Bacterial Transformation of E.Coli to Express GFP

 

Protocol Number 1: Bacterial Transformation 

On 10/20/17 my lab partner and I performed a bacterial transformation lab on E.Coli to express a Green Fluorescent Protein (GFP). This process is done by using a plasmid that codes for the GFP and inserting the plasmid by using transformation solution and heat shock. The transformation solution coats the DNA with a non-polar sheath to allow it to interact with the cell membrane of the E.Coli to allow it to pass through the phospholipid bilayer membrane of the cell which consists of mainly non polar regions. Combined with heat shock which allows the cell wall to momentarily break open, allows the most amount of uptake of plasmid. Then allowing the E.Coli to incubate allows satellite colonies to grow and express the GFP.

The picture above shows the E. Coli with the one satellite colony expressing the GFP. This result is not what we were expecting because we expected multiple colonies to grow and express GFP. We figure that most of the error came from aseptic technique. I say the because if the error lied in our ability to do the procedure we would not have seen any data.

Protocol Number 2: Inoculation of Liquid Media with Transformed Cells

 

In order to to complete the isolation of the GFP the bacteria needs to be grown in a liquid media. The liquid media is exactly like the agar plates however when making the liquid medium it lacks the protein that would normally solidify the agar.

This protocol was very straight forward in the sense that the only work to be done was to transfer the E.Coli colony from the plate to the liquid media and allow it to incubate over night with occasional breaks where the tubes need to be shook to increase the levels of oxygen.

Protocol Number 3: Concentration of Bacteria 

In this step of the eventual isolation of GFP, the main goal of this is to break open the cell wall and allow everything inside the cell to begin spilling out. This is done by taking the liquid media that has incubated the E.Coli and transfer a certain amount into a micro centrifuge tube. At this point the tubes are centrifuged at a high velocity for about five minutes. This allows the bacteria to move its was towards the bottom of the tube. This can be seen because after the 5 minutes are up a glowing green pellet can be seen at the bottom of the tube. It is at this point the liquid media is removed and a buffer is added to re-suspend the pellet to get ready for the break down of the cell wall. A drop of lysozyme is added which will initiate the break down of the cell and with that done the tubess need to be kept at a very cold temperature because freezing the tube will also aid in the process of breaking open the cell.

 

Protocol Number 4: Bacterial Lysis 

In this part of the lab the main objective is to further break open the cell wall and prepare the HIC column. The reason why ensuring the cell wall is completely destroyed is because it allows for the most amount of GFP to be collected at the end. This is because if GFP if still inside the cell wall and we “filter” using the HIC column not all of the GFP will make it to the bottom to be collected. And on top of ensuring the cell wall is open the HIC column needs to go through a “pre-run” much like a gel in gel electrophoresis needs to have a quick 2 min pre run.

 

Protocol Number 5: Hydrophobic Interaction Chromatography (HIC)

It is in this step of the protocol where the GFP and other gunk can be filtered and only leave the GFP protein to be collected. The way this works is that a series of buffers are are added to HIC column. What each buffer does is bind to different particles in the solution making it harder and harder from them to mover throughout the column. What this will eventually do for us is have all the gunk stick to white substance that is in the column while the GFP is allowed to move move much more easier that the other particles. In the end of this run there will be a little less that 1ml of pure GFP collected in a tube. The separate test tubes are conserved to do later experiments with (pertaining to concentration of a solution).

 

My Thoughts 

After completing this lab my brain was completely blown away at the idea of being able to collect a specific protein from any bacteria you want. The skills required to do this was shockingly doable and requires some knowledge of chemistry because when it gets to the point where you filter knowing how the chemistry of each particle interacts with one another is important to know. Overall I was shock at the success we had and opens my mind to all the possibilities and implications for iGEM.

Final Project Post #1

Coming into this week Hayden and I had high hopes for the success of our procedure. We came into this week done with the DNA extraction of all 10 of the samples ready to run PCR. We ran PCR on Monday and ran our gels the next day, upon completion of the gel electrophoresis there were no bands present in the gel, including the ladder, the only visual clue was the loading dye moving down the lane. This was discouraging but figured something probably went wrong during the DNA extraction because towards the end of the procedure when we should have seen a pellet there was no indication of that. Wednesday Hayden and I came in during advisement and began he process all over again this time changing the way we collected our sample. This was because the first time we did this we made sure that we got skin of the apple and zucchini and figured that may have skewed the results. Our solution to that was to get a sample deep inside the fruit/vegetable without the skin. A good indication of change is that we saw the formation of a pellet when we did not the last time (this gave us high hopes). And in order to stay on track we came in during fourth period to set up PCR again. One source of error that we might expect is that we ran out of the PCR beads and had to resort to older ones, this would mean that if bands do not show up there is a possibility that it was not our error but defective PCR beads. Thursday we are planning to run the gels and analyze our data which will bleed into Friday. We hope we will have time to prepare for the public outreach part of our project this week but will most likely happen next week and possibly not at all due to time constraints.

Chapter 24 – Cells

In this reading it was discussing fascination with cells and how cells make up all life. Throughout this reading I enjoyed the analogies that he used to describe a cell. He put it as a cell is like constructing a Boeing 77 with microscopic parts in a microscopic space and on top of that it must replicate. I liked this because it got at the point that cells are extremely complex organisms that do EVERYTHING.  A point that I enjoyed is that there are discoveries about cells that we have the opportunity to still learn about what cells can do and why they do it. Tying along to the previous point something that I didn’t like was at the end when he was tying everything together, he says that cells do all this with “random frantic action” I didn’t like this because I feel like random is not an appropriate word to use. This may due to that fact that there is not an answer to this question due to a lack of research or technology. I also found it strange that the reason why we use the name cells is because it reminded Hooke of the monks, I figured there would be a greater scientific significance to the name but hey, that’s just me. Another thing that confused me was the failed experiment that Leeuwenhoek did when he observed an explosion at close range and almost blinded himself. To that I ask that was the purpose to this experiment and what did he find. The last comment I would like to make is that when he was talking about how we lose over 500 brain cells an hour and that those cells don’t regenerate and the quote he gave after really stuck with me. That quote was, “well I guess there’s no time to waste a thought”. Over all I really enjoyed this reading because it gave really good perspective in the beginning of what a cell really is and how complex it is an also gave me a good mental model as to what a cell really looks like.