Project Proposal

Tuesday I briefly presented some of my ideas for a masters project in lab meeting. In preparation of this I wrote a project proposal document and sent it around the lab. It was very rough and incomplete, but I just wanted to get it out before the meeting. I was hoping to polish it up before putting it up here, but unfortuanetly Rosie doesn’t think it’s appropriate ( potentially very difficult to do, we’d have to invent some tools and we don’t have specialized equipment needed and even then it might not work in our organism, it’s too expansive and open ended for a masters project).  I’m moving on to other research and ideas so I’ll just post the very very rough draft here.

Davids Proposal for a Masters Project

The goal of developing a fluorescent reporter in Haemophilus influenzae is to evaluate competence gene induction and metabolism in individual cells. The competency of individual cells in a culture is thought to be heterogeneous based on observations from old congression experiments which suggest that only a minority of the cells are responsible for the majority of transformation, however these stochastic properties have not yet characterized. Additionally since fluorescence proteins can be used as a measure of metabolic activity, an indicator of the nutritional state of the cell, these reporters can be used to test the relationship between nutrition and competence. Here I will outline the research interest in single cell analysis of H. influenzae, both directly by reporters and indirectly by congression, and detail a series of experiments to test

The three main aims are as follows:

Aim 1: Characterizing congression of wildtype cultures in late log and MIV, along with in sxy-1

Aim 2: Characterizing the stochastic nature of competence, and using this information make inferences about it’s regulation

Aim 3: Determining if a relationship exists between competent cells and nutrition

Explanations of Congression

Congression is the observation that double transformants occur more often than expected, giving the interpretation that a fraction of the culture is more transformable. This is both an unexpected observation and potentially a tool for investigating the regulation of competence. This variability in phenotype is stochastic, and can result from two forms of cellular noise, intrinsic and extrinsic. Intrinsic noise is due to the random nature of chemical reactions and is exacerbated by the rarity of chemicals, which could be prominent due to haemopholis small size. Extrinsic noise is due to the differing cellular states the cells can be at, if competent cells are also more nucleotide starved perhaps cells that have recently replicated, especially if they were transferred to MIV during replication, are more competent. These two factors can be distinguished using fluorescent reporters(a). Two equivalent but differently fluorescing reporters with the same promoter will act in concert if variation is due to extrinsic factors but not if due to intrinsic factors. Interestingly stochastic effects can vary gene by gene and has been found to be correlated to function, usually interpreted as an evolutionary advantage to be heterogeneous in certain phenotypes, and this can be tested by comparing competence genes to housekeeping genes.

Nature of competence:

Approximately ten times more transformants are found after transfer to the starvation medium MIV than in late log culture. Two non mutual exclusive models with different predictions can account for this, with the truth likely falling somewhere between them.

Model 1: Ten times more cells are in MIV than late log

Prediction: Observed/Expected double transformants should increase in MIV

Model 2: Cells that are competent are ten times more competent

Prediction: Observed/Expected in MIV should be similar to late log

I’d like to test this by plating transformants. Fluorescent markers of competence induction should collaborate and resolve these predictions. I’d also like to test sxy-1 as it does not get upregulated when transferred to MIV and presumably the same proportion of cells are competent in late log and in MIV due to its stem loop being unresponsive to changes in purine abundance, and observed/expected double transformants should be the same in sxy-1 under both conditions.

Competence and Nutrition

Assuming that cells that are undergoing replication are more in need of nucleotides, and that competency is in response to nucleotide depletion, there should be some relation between the amount of DNA present in a cell and competency. Many bright stains such as syto62 are available that allow the quantification of nucleic acids in a cell. Ideally I want the use of two stains, one that binds to free nucleic pools and one that binds to incorporated DNA to discriminate between these two conditions, although I’m not aware of a stain to do this. To test the relationship of DNA availability and competency cells that have a fluorescence marker of competence can be stained along with a DNA stain, and ideally flow cytometry. This could also be tested more indirectly without the use of  fluorescent reporters, just quantifying the stains in a fluorometer and correlating with transformation frequency. Additionally I could test the relationship of constitutive metabolic rate and competence in a similar way by using a constitutive marker and a competence gene marker as constitutive markers should be dim when the cell is metabolically inactive and bright when it is metabolically active.


Although this has little to do with competence, I’m also interested in a result reported by Goodgal  that 1.6-2 cells exist in solution per colony forming unit eventually found on plates, however this wasn’t done with modern rich media.  Many of these cells may just be dead, but if they are not we are missing a large constituent of the subpopulation make up of haemophilus. A constitutive reporter gene will be able to determine if these cells are metabolically inactive

Fluorescent Reporters

Cloning fluorescent genes into a promoter of interest is a widely used technique to visualize gene expression. Candidate markers must be exceptionally bright to easily detect fluorescence in the tiny H. influenzae cells, and have a short enough lag time from translation to the maturation of the fluorophore that accurate inferences can be made about competence. Protein weight is mostly irrelevant for choosing a marker and photobleaching sensitivity depends on whether the fluorescence recovery after photobleaching(FRAP) technique will be used. In recent years new generations of fluorescent reporters have been introduced, while widely used fluorescent markers such as GFP have been optimized for use in different organisms and mutant variants constructed for use in bacteria and growth at 37 degrees. Fluorescent markers have been used successfully in haemophlius influenza[1], and modern reporters are brighter and more suitable for use in bacteria[2].

I started working with DsRED however I don’t believe it is suitable for this study due to its long maturation time, 10 hours in E.coli compared to alternative markers such as mCherrys 15 minutes[3]. I believe the best choice for studying competence in H. influenzae is mCherry due to its short maturation time and relative brightness, while td-tomato is an alternative with the greatest brightness of any spectral class but a 1 hour maturation time in E.coli[3]. If two complementary markers are needed the GFP derivative emerald would be a good choice due to its brightness, optimization for 37 degrees,  and similar maturation rate and compatibility as a dual reporter with mCherry[4]. The Venus variant of VPF is another good candidate, it has a maturation time of only 7 minutes in E.coli and nearly 80% the brightness of fluorescein, a common  and is sensitive to photobleaching which makes it ideal for FRAP

Quantifying fluorescence can be done by fluorescence microscopy, overlaying a phase contrast image and a fluorescence filter image in the same field of view and integrating fluorescence over cell area. The FRAP technique can be useful to eliminate previous fluorescence and isolating only the induction of competence.  A flourometer can be used as a control for methodology by checking if changes between conditions are proportional with those seen with microscopy.

I’m concerned that haemolipus small size will make fluoresnce hard to detect in individual cells. In E. coli 30 copies of GFP need to be present to easily see a cell. However recent advances both in markers and detection methods allow for the visualization of single fluorescent molecules. Proteins such as td-tomato are much brighter than GFP and are shifted further away from the cells background autofluoresccnce(due mostly to flavin).  Techniques like cloning a membrane targeted sequence to the marker and using a short excitation pulse(strobe photography) also help greatly with visualization. A high powered argon laser and a cooled ccd camera with high quantum efficiency are needed for effective microscopy. Flow cytometry is superior to fluorescence microscopy for detection and quantification.

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I would like to thank Sunita for her continual support, advice and knowledge, Josh for his discussions and especially developing the models for MIV, and Rosie for her helpful comments and questions on previous write-ups.

(This is a very rough section I’m not sure to include/if it fits)

Does Nutrition quench Competence?

Goodgal 1961 reports that when cells are grown in media with DNA they are less transformable compared to cells grown without the added DNA. I’d like to repeat these experiments, and again see if sxy-1 is affected differently than wild type. Transformation frequency peaks around 100 minutes in MIV and then diminishes(?). Perhaps this is because if nutrient starved competent cells don’t find DNA they enter a post competent state where they are unable to transform but can still be rescued when transferred to rich media. If all cells in a culture can be made to be equally competent, and DNA ‘quenches’ competence, the observed/expected ratio of double transformants should be below one.

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