The first step in developing a robust system for the production of recombinant GFP, requires knowledge of the growth characteristics of the host organism; in our case Escherichia coli. The standard approach to obtaining this information is to measure the rate of growth of the organism under defined conditions. It may also be useful to determine to what extent factors such as temperature, growth medium composition and pH, vessel configuration, aeration rates etc., influence the yield of cells. The method we shall use, and indeed you have developed yourselves (with a little bit of help), is to monitor growth over time using a spectrophotometer to accurately measure the turbidity (cloudiness) of a liquid culture at a set of well defined time points.
The starting point and the end point of the experiment should be considered in planning your schedule. This is where you need to think carefully about managing your time and organising materials and equipment. I wont give away the method completely, but you might want to think about the following elements of the experiment.
1. What volume of culture should I use? Why?
2. What is the best inoculum to use (loop or defined volume) and how could I determine this empirically?
3. What is the likely time taken for sufficient numbers of cells to populate the culture tube making detection possible. (Hint, you already have your own data on plate and broth cultures). Discuss this with your colleagues.
4. What sample volume is ideal for recording time points?
5. What solution should I use as a "blank"
6. Why did I suggest placing your first sample on ice today?
7. Are there any consequences of depleting the culture during the experiment?
8. What are the growth characteristics of bacterial cultures that have been described in class or on the Internet (e.g. Wikipedia)
9. Should I make single, duplicate or triplicate measurements, or maybe team up with colleagues? Pros and cons?
10. How should I record and plot my data?
11. What should I do after plotting my data?
12. What do other students' data look like and can I rationalise the results?
Key words Growth curve, E.coli, temperature, pH, aeration, spectrophotometry, blanks, culture, medium, planning, time management, organisation
This week has seen us return to bench work and as you know we are set on a course to purify our own Green Fluorescent Protein before Christmas. Over the last few days, you will gave become used to my sarcasm over your economic approach to the use of adjectives? One of the most important aspects of Scientific endeavour is the ability to make careful observations....and record them concisely and quickly. In addition, you should write up your methodology in a way that will enable you (and anyone else)to obtain the same set of experimental results. Finally, you must label all of your samples carefully, so that you can locate your plate or tube after a day, a week or a year, if necessary.
As you will have noticed I pushed you to describe the plates above (well your own poured and inoculated plates) in detail. I was looking for comments on
Percentage coverage of the plate
Distribution of the colonies
Shapes and sizes of the colonies
Colour and appearance of the colonies
Sizes of (and shape) single colonies (did you use the magnifier?)
Number of colonies estimated by "sectoring" the plate
Did the culture have any characteristic smell?
Was there any evidence for contamination?
You can read more at one of my earlier Blog Posts http://utcinnovationlabs.blogspot.co.uk/2014/06/elementary-my-dear-watson.html
Key words Observation Recording findings
This week we have started BLASTing again in Y12 classes and by the end of the first weekly sessions, all of you will have gone through the basic steps. I walked around at the end of each session yesterday and checked that you had all worked through the CFTR primary structure search and then your own searches looking at the human h(a)emoglobin alpha chain sequence similarities across the species. I wanted to just add a reminder for the two key sites and in particular to encourage you all to sign up for an NCBI account: it will store all of your searches and will be something to show to future employers or University interviewers!
NCBI (and remember to use BLASTp)
PubMed (and remember to select Proteins, from the drop down menu)
A second tip at this stage is to add terms to refine your search. As I said a thorough grounding in classical languages helps with BLAST. So if you want to compare a sequence from man (eg Histone) add homo. If you are looking for a bacterial DNA Polymerase add coli. This will help you select the correct "hit" from the long list. Finally, check the number of amino acids in the sequence you are looking for. This is given in the summary information for each hit. So a 13 aa sequence is not likely to be correct for CFTR which is nearly 1500 aa (use wikipedia if in doubt to find more info about your query sequence). We shall continue with the introduction-search-discuss-search again sessions over the next few weeks.
Key Words BLAST, Primary structure, sequence alignment, NCBI, PubMed
The first step in developing a robust system for the production of recombinant GFP, requires knowledge of the growth characteristics of the host organism; in our case Escherichia coli. The standard approach to obtaining this information is to measure the rate of growth of the organism under defined conditions. It may also be useful to determine to what extent factors such as temperature, growth medium composition and pH, vessel configuration, aeration rates etc., influence the yield of cells. The method we shall use, and indeed you have developed yourselves (with a little bit of help), is to monitor growth over time using a spectrophotometer to accurately measure the turbidity (cloudiness) of a liquid culture at a set of well defined time points.
