COVER PAGE

Experiment 1

This experiment

hypothesises that the pathogen-secreted effector molecule (27 kDa) protein is secreted after the pathogen binds to gastrointestinal epithelial cells (Chaffanel, et al., 2018). To investigate this, we would make use of the flow cytometry experiment to analyse whether this binding association is paramount for the release of the effector molecules. The pathogen used for this experiment is the Streptococcus salivarius bacteria. This bacterium is well known to induce the production of 27 kDa protein in the IECs. First of all, we would culture the epithelial cells in L-RWN conditioned media (Dussen, Sonnek, & Stappenbeck, 2019). You ought to have three agar plates having the Gastrointestinal epithelial cells grown on a media. The first agar plate will have no Streptococcus

salivarius inoculum added to it. It serves as a negative control for the experiment. The second agar plate contains the Streptococcus salivarius bacteria but with a binding-inhibitor complex X. This inhibits binding of the pathogens to the IECs. The third agar plate contains the IECs together with the pathogen and no inhibitor. The above plates are incubated for one hour.

CONCLUSION

The agar plates are

retrieved and by use of the Flow cytometry technique, to detect the production

of the pathogen secreted effector molecule by the IECs. It is expected that

none of the 27 kDa proteins should be produced from the first agar plate since

there were no pathogens introduced into the culture. There should be a

production of 27 kDa protein from the 3rd plate. The second plate contains the pathogen and the IECs, but no binding occurs. If any of the 27 kDa proteins are detected, we would disregard the hypothesis and conclude that binding of the pathogen to the IECs is not paramount for the secretion of this molecule. However, if none of the pathogen-secreted effector molecules is detected, we would uphold the hypotheses. It is expected that the 27 kDa proteins should be produced from this agar plate which allows for binding of the pathogen to the IECs.

Experiment 2

Three different culture

mediums are to be used in this experiment. The first culture medium will

contain L-RWN medium is inoculated with Intestinal epithelial cells (Dussen,

Sonnek, & Stappenbeck, 2019). The second and third cultures were used for culturing neuron cells. Each of the plates

is incubated with its respective cell types for one week. Once grown each of

the plates are retrieved. The 27 kDa proteins are then introduced into each of

the different media at a concentration of 10g/ml for the agar plates 1 and 2.

The 27 kDa protein is not introduced in the third agar plate. It is set up as a

negative control for the experiment. The plates are incubated for 24 hours. A

primary antibody specific for binding to the pathogen-secreted effector

molecule is introduced. The plates are incubated for 30 minutes. The plates are

retrieved, and the antibodies washed to remove all the unbound antibodies. A

secondary antibody (fluorescently tagged)) is then introduced to

each of the agar plates. The plates are incubated for another 30 mins. All the

plates are washed to remove the excess and unbound secondary antibody. The

remaining cells are fixed using 3% paraformaldehyde. By use of a fluorescence

microscope, each of the agar plate is observed and any fluorescence from any of

the plates noted.

CONCLUSION

From the hypothesis, the

first sample should not produce any inflorescence. The first plate consisted of

Intestinal epithelial cells. If any inflorescence is detected in this plate,

the hypothesis would have been proven wrong. The second plate contains neuron

cells. These cells should be able to take in the 27 kDa proteins and have them

localised in their cytoplasm. It should be able to emit fluorescent rays to

confirm this. The third plate contains neuron cells; however, these were not

incubated with the effector molecules. Thus, the free unbound primary antibody

should have been washed off. The third plate is the negative control for this

experiment.

Experiment 3

For this experiment,

three different mice are to be used. Three healthy mice are acquired separately

placed in 3 different (carton boxes). The *boxes* should be similar in size

with same internal conditions. This reduces on the effects of the environment

on expression of the genes. The first mouse is injected with Streptococcus salivarius bacteria at a

concentration of 100mg/ml. The second mouse is injected with the same bacteria

at a concentration of 10mg/ml. To act as our negative control, the third mouse

is not injected with any pathogen. They are then taken back to the laboratory

and fed for three days. The mice are then retrieved, and each diagnosed for the

level of dementia-like symptoms expressed. The first mouse is observed and

graded as either mild, moderate or severe. The same is done for the second

mouse and the third mouse.

CONCLUSION

This hypothesis was set

to test whether the presence of the pathogen-secreted effector molecules

influences the level of expression of the xyzA gene. The xyzA gene was verified

by grading observations made on the mice about the dementia-like symptoms

expressed. The first mouse is expected to show a severe level of expression of

the dementia-like symptoms. This mouse was injected with the highest

concentration of pathogen injection; hence it should have the highest level of

xyzA gene expression, and as a result, the symptoms should be more severe. The

second setup had the mouse injected with a low concentration of Streptococcus salivarius bacteria. The

result should be moderate to mild since the level of expression of the xyxA

gene would also be minimum. The third mouse is expected to show no

dementia-like symptoms. There was no injection with a bacterium. Hence no

effector molecules should be in the neuron cells and as a result minimal

expression of the xyzA genes.

Works Cited

Chaffanel, F., Bourgoin, F. C., Soligot, C., MOunira, K., Stephanie, B., Sophie, P., . . . Nathaniel, L. B. (2018). Surface proteins involved in the adhesion of Streptococcus salivarius to human intestinal epithelial cells. Springer, 2851-2865.

Dussen, K. L., Sonnek, N. M., & Stappenbeck, T. S. (2019). L-WRN conditioned medium for gastrointestinal epithelial stem cell culture shows replicable batch-to-batch activity levels across multiple research teams. ELSELVIER, 37.