02/20/19

Rationale:

to acquire the fully annotated genome of Napoleon B

Procedure:

1. reviewed the annotated genome with the class and we were instructed to make possible changes
2. Talked about poster design and the important elements of  making a scientific poster.

Results:

3 corrections were made to the genes of Napoleon B and all genes were reviewed.

Conclusion

the genome is now fully annotated and ready for review.  there are many gaps in the genome , which is very peculiar. something can be looked into

Future steps:

work on individual research projects

02/18/19

Rationale: to acquire the fully annotated genome of Napoleon B

Procedure:

1. DNA master was opened and Napoleon B was opened in DNA Master.
2. Gene 44 was reevaluated
3. NCBI was used to blast the gene and acquire genes in the database that matched with the query gene, which was used to find the possible similarity and function of the query gene
4. Genemark was used to check the coding potential covered by the gene and the possible changes that could be made if possible to acquire the longest ORF possible.
5. Phagesdb was used to blast the gene and acquire genes in the database that belonged to other phages that matched with the query gene, which was used to find the possible similarity and function of the query gene
6. HHpred was used to predict the protein structure of the gene product, which was run through its database (Pfam and COG)for a possible matches with other proteins, which could be used to predict the function of the protein.
7. Phamerator was used to compare the genome of napoleonb to other phages in the same category to compare syntony of the genes.
8. phage notes was used for input of all the required information correcting the gene annotation.
9. After, the abstracts of group members were compared and one sample abstract was submitted.

Results:

Gene 44 annotation:

SSC:30372 – 30815, CP:Yes, SCS:Both, ST:SA, BLAST-Start:Aligns with Circum gp46 NCBI BLAST q44:s1 1 6E-106, Aligns with Circum gp46 PhagesDB BLAST q44:s2 1 9E-84, Gap:8bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 3.102 -3.355 No, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF, SIF-Syn:NKF

Conclusion:

gene 44 has no known function.

Future steps

select abstract for scholars day submission

02/11/19

Rationale:

to acquire the annotated genome of  napoleon b,

Procedure:

1. opened  DNA master
2. Opened FastA file for napoleon b using the following steps, File>open>FastA multiple sequence file> Choose NapoleonB.FastA
3. clicked export on window with the extracted file and then choose Create sequence from this entry only.
4. opened frames and went to DNA> Frames.
5. then clicked ORF on the new window to see the highlighted genes.
6. Gene 69, 70, and 71 were annotated
7. NCBI was used to blast each gene and acquire genes in the database that matched with the query gene, which was used to find the possible similarity and function of the query gene
8. Genemark was used to check the coding potential covered by each gene and the possible changes that could be made if possible to acquire the longest ORF possible.
9. Phagesdb was used to blast each gene and acquire genes in the database that belonged to other phages that matched with the query gene, which was used to find the possible similarity and function of the query gene
10. HHpred was used to predict the protein structure of the gene product, which was run through its database (Pfam and COG)for a possible matches with other proteins, which could be used to predict the function of the protein.
11. Phamerator was used to compare the genome of napoleonb to other phages in the same category to compare syntony of the genes.
12. phage notes was used for input of all the required information for genomic annotation.

Results:

the function declared for all three genes was “no known function”.

the BLAST results yielded genes of unknown function, same was concluded for HHpred.

Conclusion

these three genes have no known function and do not seem to have any significance as of now. nothing further can currently be concluded.

Future steps

Finish annotation by annotating gene 72.

Rationale:

to increase focus on certain genes and learn to annotate genes

Procedure:

1. open  DNA master
2. Preferences >Local settings >New Features
3.  Type the following template command in the the “template to be installed box” :” SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST: SIF-HHPred: SIF-Syn .
4. check the “insert template into notes during Auto Annotation” box. Then Apply>OK
5. Open FastA file for elesar using the following steps, File>open>FastA multiple sequence file> Choose Elesar.FastA
6. click export on window with the extracted file and then choose Create sequence from this entry only.
7.  to see the frames, go to DNA> Frames.
8. then click ORF on the new window to see the highlighted genes.
9. this is where time for lab ended.

Results:

auto annotation identified in 65 ORFs, of which 12 were reverse ORFS and 53 were forward ORFs, of which ORF 19 waas the longest.

Conclusion:

Reverse ORFs seem to be the most uncommon in the genome. more interpretation may be later required due to lack of porper knowledge as of now.

Future Steps:

the next lab will involve more work identifying genes and find gaps and overlaps between the auto annotated genes. It may involve identification of possible things that autoannotation missed.

1/16/19

Rationale:

To install and use DNA master to  auto annotate and analyse the genome of phage elesar.

Tools Used:

• Personal Laptop
• DNA master

Procedure:

1. DNA master was downloaded and installed onto personal laptop.
2. The following steps were followed to go to local settings, File > Preferences> Local settings> Directories
3. Archive file settings were changed to a location where it would be easier to find
4. The color settings for tRNA, tmRNA and ORF are changed and all the changes are applied
5.  the elesar genome file was opened in DNA master using the following, File>Open>FastA multiple sequence file
6. the following steps were followed, Export> Create sequence from this entry only>Genome>Auto Annotate
7. this is where the day ended.

Observations:

The operation of DNA master is difficult and will take a great deal of practice. Elesar has 66 genes, of which one was tRNA and the rest are ORF genes.

Results:

successful auto annotation was performed on the genome sequence. ORFs on the gene sequence were marked as shown above.

Conclusions and Future Steps:

Currently, no conclusions can be drawn from this data due to the lack of the knowledge to do so. more practice and augmentation is required to better work with this software.

11-28-18

Objective:

To pick 3 plaques and make a plaque assay from the extracted phages.

Pre-Lab Observation:

The plaque assays prepared on 11-26-18 seemed to have a regrowth in Arthrobacter and can therefore not really be used. it is difficult to state whether plaques had formed. Because all the lysate was used, another plaque assay cannot be made. therefore, in a final attempt, an older plate was acquired and plaques were picked.

Procedure:

1. The aseptic zone was set up.
2. 3 plaques were picked from the old plaque  assay and the phages were deposited in a microcentrifuge tube with 30μl of phage buffer.
3. 30 μl of this phage extract was deposited in 0.5 ml of arthrobacter and was allowed to enrich for 15 minutes.
4. 1 TA mixture was prepared for three plates.
5. 6 ml of LB broth was transferred to a conical vial.
6. 67.5 μl of CaCl2 was added to the conical vial.
7. After the lysate was allowed to enrich for 15 minutes, 7.5 ml of 2X TA was added to the conical vial.
8. 4.5 ml of the TA mixture was added to the arthro tube.
9. The contents of the tube were then poured onto the agar plate.
10. 4.5 ml of the 2X TA mixture was plated on an agar plate for a control.
11. the plates were allowed to solidify for 15 minutes.
12. the plates were then placed in the incubator.

Analysis and Conclusion:

This is the final attempt. If the plate is webbed, it can be flooded and process for DNA extraction can be started.

11/26/18

Objective:

• Make a plaque assay from a picked plaque

Pre-Lab Observation:

The spot test prepared on 11/14/18 resulted in a weak titer or an absolute lack of plaque. Another group member was assigned to extract lysate from the same soil and make a plaque assay. plaques were picked from the plaque assay in this lab in an effort to restart purification.

Procedure:

1. The aseptic zone was set up.
2. A plaque was picked from the group members plaque  assay and the phages were deposited in a microcentrifuge tube with 50μl of phage buffer.
3. 50 μl of this phage extract was deposited in 0.5 ml of arthrobacter and was allowed to enrich for 15 minutes.
4. 1 TA mixture was prepared for three plates.
5. 6 ml of LB broth was transferred to a conical vial.
6. 67.5 μl of CaCl2 was added to the conical vial.
7. After the lysate was allowed to enrich for 15 minutes, 7.5 ml of 2X TA was added to the conical vial.
8. 4.5 ml of the TA mixture was added to the arthro tube.
9. The contents of the tube were then poured onto the agar plate.
10. 4.5 ml of the 2X TA mixture was plated on an agar plate for a control.
11. the plates were allowed to solidify for 15 minutes.
12. the plates were then placed in the incubator.

Analysis and Conclusion

50 μl of the extract was used to possibly get a webbed plate. the probability of such an outcome is small. due to the lack of time, this would be the last chance for possibly getting to DNA extraction, which seems unlikely at this time.

11/07/2018

Objective:

Pick plaques and make plaque assay for the extracted phage and 10^-1 dilution of the lysate.

Pre Lab Observation:

The control plate from the plaque assays from 11/05/18 were contaminated and therefore reliable plaque assays were not obtained.

Procedure:

1. 30 plaques were picked from 3rd purification run and deposited in a microcentrifuge with 100μl of phage buffer.
2. After phages are mixed in the microcentrifuge tube, 10 μl of the extracted phage solution is transferred to a microcentrifuge tube with 90μl of phage buffer to acquire a 10^-1 serial dilution.
3. One top agar mixture was prepared for 5 plates.
4. 10 ml of LB broth was added to a conical vial
5. 112.5 μl of CaCl2 was added to the conical vial.
6. Two 0.5 ml of arthrobacter samples were enriched with the extracted phages and the 10^-1 dilution for 15 minutes.
7. 12.5 ml of 2X TA was added to the conical vial.
8. 4.5 ml of the TA mixtures was added to each enriched sample.
9. the samples and the mixture were then plated on two agar plates.
10. 4.5 ml of TA mixture was plated on another agar plate for a control plate.
11. after 15 minutes, all the plates were placed inverted in the incubator.

Analysis and Conclusion:

Most likely due to the contamination, the plates prepared on 11/05/18 were negative and a webbed plate was not produced. Due to the lack of lysate, another purification was done to acquire more phages. New titer will have to be calculated and a webbed plate will have to be prepared during the next lab.

10/29/18

Objective:

• Collect a new soil sample
• Take pH of soil
• Calculate % water

Procedure:

Collection:

1. collect soil in a Ziploc bag
2. measure the circumference of the tree trunk 137cm from the ground
3. measure the longest diameter and shortest diameter of the tree canopy and take its average.
4. complete metadata survey

pH:

1. a small amount of soil was added to the pH vial
2.  water was added to the vial until the level reached the top of the vial
3. the vial was shaken for 10 seconds and then allowed to rest for 120 seconds
4.  after 120 seconds, the pH paper was immersed in the vial for 45 seconds
5. the pH was determine using the color scale on the chart in the pH paper dispenser.

% water

1. weighing boat was weighed on the scale ( g)
2. some soil was added onto the boat and weighed
3. the difference of the final weight ( boat and soil ) and initial weight (boat) was taken to find the weight of the soil.
4. the soil was placed in the fume hood for 48 hours, after which it will be weighed again.

Analysis and Conclusion:

The pH of the soil acquired was 6.8.

10/24/18

Objective:

• Test for the presence of phage clusters in lysate of soil sample C using PCR and Gel Electrophoresis.

Calculations:

Total Volume= 35 ml

Density of Et Br= 10μg/μl

For 2% agarose = 0.02 X 35= 0.7g

For 0.5% Et Br= 0.005 x 35 x 10= 1.7 μg

Procedure:

1.  35 ml of TBE water was poured into a flask.
2. 0.7 g of Agarose powder was added to the flask
3. The flask was microwaved until the solution bubbled. the flask was taken out  and shaken. Because clumps of powder still remained in the flask, the flask was microwaved and shaken again until all the powder had dissolved.
4. The solution was allowed to cool until it was warm, at a temperature of about 55°C.
5. 1.7μl of Et Br was added to the flask.
6. The solution was then poured onto a electrophoresis apparatus plate (with two dams and combs) and the gel was allowed to solidify.
7. the comb and dams were carefully removed once the gel solidified.
8. The plate was then placed into the electrophoresis apparatus.
9. The apparatus was filled with TBE water until the gel was completely submerged.
10. 5μl of the DNA ladder was put in one of the wells in the gel.
11. 10 μl of  the contents of the three green centrifuge tubes prepared on 10/22/18 were placed in three wells in the gel.
12. The apparatus was connected to the power source and the voltage was set to 100V.
13. the gel was allowed to rest in this electric field for 45 minutes.
14. After 45 minutes, the gels were removed from the apparatuses and placed into a machine that emits UV light, analyzes and records the readings on the gel.

Analysis and Conclusion

My Sample ( along with lab partners)control

After analysis of the results from the gel electrophoresis, this soil does not seem to contain any phages and therefore a new soil sample will now be required. There may also be a possibility that adjustment may be required due to varying quality of the result due to use of different Taq polymerases. this may also be further tested. the verity of the PCR will also be tested with the use of lysate samples that contain phages for sure