Tyler+T.+(Springer)

= = Tyler - great work. I appreciate your attention to detail and understanding of the concepts that are in VDS. - DR. B 121214 = = =Weeks 14 & 15=

Control Ligand Library - 11/25/14

A control ligand library was established containing 10 positive control ligands and 5 negative control ligands, including aspirin. The library was then docked into the active site using GOLD molecular docking software and ranked according to GOLD fitness score.



= =

Analysis:
A control ligand library was established in order to ensure the validity of the GOLD molecular docking software. Positive control ligands were determined using the Binding Database. Compounds that were found to be known inhibitors of protein tyrosine phosphatases in other bacteria, such as //Mycobacterium tuberculosis//, were selected as positive controls. These compounds were expected to bind strongly with the active site and achieve overall higher GOLD scores. Negative control ligands were determined using the ZINC database. Negative control ligands were selected at random from a list of compounds with similar physical and chemical properties to the positive control ligands. Because there was an overall difference between the average scores of positive and negative control ligands, we can move forward with the virtual screening of the ChemBridge Diversity library and other libraries knowing that the GOLD software is valid.

Restriction Enzyme Digest - 12/1/14

Restriction enzyme digest was performed a second time on the pNIC-Bsa4 DNA plasmid using BsaI-HF in order to remove the sacB gene and provide a location for the insertion of the gene of interest, //lmo1800//.

= =

Analysis:
The RE digest protocol was performed a second time on pNIC-Bsa4 because we had gone through two cloning attempts without success. We determined that too much time had passed between the time at which we had cut the pNIC and when we attempted the cloning protocol. We figured that freshly cut pNIC may be the solution to our cloning problem as different ratios were not proving successful. The RE digest protocol was considered successful because bands were observed at approximately 1.5kb and 0.5kb, the length of the plasmid and sacB gene, respectively. Interestingly enough, there were two other bands present that could not be explained. One possible explanation may concern the fact that we were forced to use CutSmart buffer rather than the 10X reaction buffer called for by the protocol. The NEB website said that this substitution should yield the same result. The next step will be to perform the cloning procedure for a third time using the freshly cut pNIC-Bsa4.

Cloning - 12/2/14

Cohesive end generation was first performed in order to create sticky ends on both the pNIC-Bsa4 vector and the insert so that complementary base pairing could occur. The vector and insert were then annealed so that hydrogen bonding could occur. Finally, the vector and insert were used to transform DH4a //E. coli// cells. This was the third time the cloning procedure has been performed, though this time, freshly cut pNIC-Bsa4 was used.



Analysis:
After two failed cloning attempts, we decided to redo RE digest and use freshly cut pNIC-Bsa4 as the accepting vector. This proved to be successful, yielding a positive clone in each of the three plates. A positive clone indicates that the sacB gene has successfully been removed as the cells can grow in an environment of sucrose and that the pNIC plasmid is present conferring resistance to kanamycin. Now that positive clones have been created, they need to be sent to sequencing in order to confirm the DNA sequence. Once the sequence has been confirmed, a master plate can be created and LmPtp can be overexpressed and purified for use in assays.

=12042014- Keep up the good work, outstanding job!= =Weeks 11, 12, & 13= PCR Cleanup - 11/6/14

PCR cleanup was performed to obtain a pure sample of pNIC-Bsa4



Analysis:
PCR Cleanup yielded a pure sample of pNIC-Bsa4 plasmid at a concentration of 30.2 ng/uL. Though this concentration is relatively low, we plan to use it to clone our gene.

RE Digest of pNIC-Bsa4 - 11/7/14

pNIC-Bsa4 was cut using the restriction enzyme BsaI-HF to remove the sacB region from the plasmid. 55.83uL of the plasmid was used, and all other volumes of reagents were tripled. The concentration of the results were determined using the Nanodrop spectrophotometer and found to be 72.6ng/uL.





Analysis:
RE digest was performed in order to remove the sacB gene from the pNIC-Bsa4 plasmid so that our gene of interest could be inserted. Now that the plasmid has been cut, we plan to perform cohesive end generation on both the vector and insert to promote hydrogen bonding and insertion.

Homology Model - 11/17/14

A homology model was created from a protein tyrosine phosphatase in //Mycobacterium tuberculosis// using ICM because no crystal structure is available for Lmptp.



Analysis:
A pairwise alignment provided a 26% similarity between Lmptp and the PtpB in //M. tuberculosis//. This was unfortunately the highest similarity that we could find in current literature. It was also noted in a paper that this particular protein had been used as a homology model in the past for Lmptp. The MolProbity analysis provided a poor outlook, though we will use this in an attempt at virtual screening.

Cohesive End Generation/Annealing and Transformation - 11/18/14

Cohesive end generation was performed in order to create ends on both the vector and insert that could complementary base pair to one another. The vector and insert were annealed in order to create hydrogen bonds, and then the cloned gene would have then been used to transform DH5a E. coli cells.





Analysis:
No growth was observed on either of the plates, indicating that cloning was unsuccessful. Possible sources of error include incorrect ratios, contamination, or even failed RE digest. We will complete the cloning procedure again in an attempt to get a positive result.

Cohesive End Generation/Annealing and Transformation - 11/21/14

The same protocol was repeated, though different ratios were included in the second trial.







Analysis:
Once again, there was no observed growth indicating that cloning had failed. Possible sources of error include incorrect ratios, contamination, or even failed RE digest. Because RE digest was performed several weeks ago, we intend to retry the RE digest procedure before trying cloning once more. =Weeks 8, 9, & 10= Secondary PCR - 10/17/2014

Previously ordered forward and reverse primers were added to the mix with 1.0uL of primary PCR mix. Times and temperatures used were identical to those of primary PCR.

Cycling Conditions: 98 degrees Celsius - 30 seconds 98 degrees Celsius - 7 seconds 62.5 degrees Celsius - 20 seconds 72 degrees Celsius - 25 seconds 72 degrees Celsius - 2 minutes 4 degrees Celsius - infinite



Analysis:
Secondary PCR failed. There were no dark bands observed which would have indicating successful strands of cohesive DNA. Sources of error include the annealing time and temperature, which we intend to alter. For the next trial, we intend to use an annealing temperature that is 5 degrees Celsius less than the melting point of the primers.

Secondary PCR - 10/21/2014

Once again previously ordered forward and reverse primers were added to 1.0uL of primary PCR mix. The annealing temperature was changed to 54.5 degrees Celsius which was 5 degrees Celsius less than the melting point of the primers.

Cycling Conditions: 98 degrees Celsius - 30 seconds 98 degrees Celsius - 7 seconds 54.5 degrees Celsius - 20 seconds 72 degrees Celsius - 25 seconds 72 degrees Celsius - 2 minutes 4 degrees Celsius - infinite



Analysis:
Once again, secondary PCR was a failure. Even though we used an annealing temperature of 54.5 degress Celsius, we saw no bands to indicate a successful result. We are beginning to believe that this is due to insufficient yields from primary PCR. Though we had a successful primary PCR, it was not a very strong smear. We plan to redo primary PCR, using more of each element, in order to yield a stronger result. Hopefully this will lead to a successful secondary PCR.

Primary PCR - 10/28/2014

Primary PCR was repeated in order to achieve a stronger result. An additional 1.0uL of oligo mix and an additional 0.5uL of Q5 polymerase were added to the reaction mixture.

Cycling Conditions: 98 degrees Celsius - 30 seconds 98 degrees Celsius - 7 seconds 62.5 degrees Celsius - 20 seconds 72 degrees Celsius - 25 seconds 72 degrees Celsius - 2 minutes 4 degrees Celsius - infinite



Analysis:
This trial involved repeating primary PCR, but using an additional 1.0uL of oligo mix and an additional 0.5uL of Q5 polymerase. Primary PCR was successful as we observed a very strong smear in Lane 3. We will use the resulting mix in secondary PCR.

Secondary PCR - 10/30/2014

Secondary PCR was redone using the results from the most recent, stronger primary PCR. The annealing temperature was kept constant at 54.5 degrees Celsius.

Cycling Conditions: 98 degrees Celsius - 30 seconds 98 degrees Celsius - 7 seconds 54.5 degrees Celsius - 20 seconds 72 degrees Celsius - 25 seconds 72 degrees Celsius - 2 minutes 4 degrees Celsius - infinite



Analysis:
We repeated secondary PCR using the same conditions as before. This time, we observed a successful result. There was a dark band in Lane 3 indicating a successful run. This is indicative of cohesive strands of DNA which can now be amplified using PCR Squared. Because no other conditions were changed, we attribute the successful run to redoing primary PCR with a more successful result.

PCR Squared - 10/31/2014

PCR Squared was used to amplify the cohesive gene that was a product of secondary PCR. The cycling conditions used were identical to those of secondary PCR.

Cycling Conditions: 98 degrees Celsius - 30 seconds 98 degrees Celsius - 7 seconds 54.5 degrees Celsius - 20 seconds 72 degrees Celsius - 25 seconds 72 degrees Celsius - 2 minutes 4 degrees Celsius - infinite



Analysis:
A dark band around 900bp is indicative of a successful PCR Squared run. The gene was amplified successfully. Next, we will repeat PCR squared in order to produce more of the lmo1800 gene which can then be cloned into the pNIC-Bsa4 vector. Shortly, we will begin cutting pNIC using BsaI in order to prepare it for insertion of the gene. = =

=Weeks 6, 7, & 8= 10232014- good job, try to include better images from your PCR results Primary PCR of //lmo1800// oligos

Oligos were mixed together in a tube and then primary PCR was performed using Q5 DNA polymerase and the standard NEB thermocycler cycling conditions.



Analysis:
PCR was performed twice and both times resulted in failure. We should have seen a streak in lanes three and four, which would have indicated a mixture of the successfully linked gene as well as other fragments of DNA. There was no streak or bands indicating that the oligos had not been linked by the DNA polymerase. Standard NEB conditions were used in the procedure. The first attempt at primary PCR failed most likely because we used a 1:2000 dilution of the Q5 rather than a 1:2 dilution. The second attempt also failed, most likely because the temperature of the annealing step is not correct. For both trials, it was set to 58 degrees Celsius. This difference in temperature resulted in the DNA primers not properly attaching to the DNA template. The next step will be to redo primary PCR using an annealing temperature closer to the melting point of our oligo primers.

Midi-prep

The midi-prep procedure was performed in order to lyse the bacterial cells and purify our sample of pNIC-Bsa4 DNA plasmid.





Analysis:
The midi-prep procedure was performed twice. Initially, we obtained a yield of 26 ng/uL using Monica's cells. This was not sufficient for cloning, so we performed the procedure again, this time using my cells. The second time, we obtained a yield of 40.3 ng/uL, which is still not as high as we would like it to be, though it might suffice for cloning. pNIC-Bsa4 is a low copy plasmid by nature. The second time we performed the procedure, we used half of the volume of buffer in order to obtain a more concentrated sample. Sources of error include contamination, errors in measurement, and errors in the time sensitive portions of the procedure. The next step involves using restriction enzymes to cut the sacB portion out of pNIC-Bsa4 and inserting our own gene within the plasmid.

Tail Primer Design

Forward and reverse primers were designed for the //lmo1800// gene so that it could be successfully replicated within the accepting vector, pNIC-Bsa4. NEB Cutter was used to determine where the plasmid would be cut using the restriction enzyme BsaI.






 * FORWARD PRIMER SEQUENCE**: TACTTCCAATCCATG AAAAACTGGGTTAAAGTTAC


 * REVERSE PRIMER SEQUENCE**: AGAAAGCTTACCTGTACTAACAGTAAAGGTGGATA

Analysis:
The forward and reverse primers created will be used during secondary PCR in order to create a complete version of //lmo1800// that has the potential for insertion into the plasmid vector. Once BsaI has cut the plasmid in the locations indicated above, the gene of interest can then be inserted by utilization of the sticky ends created by the enzyme.

=Weeks 3, 4, & 5= My First PCR of pGBR22

A master mix of DNTPs, M13F and M13R primer, buffer, and water was first created. Three dilutions of pGBR22 were then made and added to equal amounts of master mix. One final tube was made with no plasmid as a control. Taq polymerase was then added and the tubes were run through agrose gel.



Analysis:
PCR was unsuccessful. Clear bands did not appear for any of the plasmid samples. This could have been a result of the low initial concentration of DNA plasmid, 18.8ng/mL, or possible contamination. Also, the temperatures at which the PCR was run and the duration of each step could have affected the results. The next step would be bacterial transformation with the amplified plasmid.

Bacterial Transformation (Days 2 & 3)

One colony from the transformed plate of DH5a E. coli bacterial cells was selected and grown in LB + KAN liquid media for 16 hours in a shaking incubator at 37 degrees Celsius. The solution was then centrifuged at 4 degrees Celsius at 6000 x g for 15 minutes. The resulting pellets were then stored at -20 degrees Celsius.



Analysis:
The culturing of a single colony in LB + KAN was successful. The solution incubated overnight was cloudy, indicating bacterial growth, and two substantial pellets resulted after the solution was spun down. The bacteria have been transformed as they can now survive in an environment with KAN. This can be confirmed with protein characterization as the next step.

RE Digest

The restriction enzymes EcoRI, PvuII, and EcoRI + PvuII were used to cut the DNA plasmid pGBR22 in preparation for the gene to be cloned. A combination of of buffer, enzyme, water, and plasmid were incubated at 37 degrees Celsius to allow the restriction enzymes to work and then heat shocked to stop the process. The results were then run through an agrose gel to determine whether or not the plasmid had been cut in the correct places.



Analysis:
RE digest was unsuccessful. There are no clear bands in lanes 4 through 6, indicating that the plasmid had not been cut in the correct places. Sources of error include the addition of the restriction enzyme. I do not think that it was actually added to the plasmid as it was such a small amount. Has this been successful, the plasmids would have been ready for gene insertion.

982014- Nice work, but don't forget both pictures of your Nanodrop next time. = = =Weeks 1 & 2= Bacterial Transformation (Day 1) - 9/2/2014

We began by using the Nanodrop Spectrophotometer to determine the concentration of the unverified pNIC-Bsa4 DNA plasmid. Because the 260/280 and 260/230 values were approximately 1.8 and 2.1, respectively, the sample is considered relatively pure. We performed the transformation by adding 50ng (7.59 uL) of the plasmid to 25 uL of the E. coli bacteria. The bacteria and plasmid were then mixed with SOC media and plated onto LB+KAN. One plate received 10 uL while the other received 50 uL of the bacteria. The plates were then allowed to incubate at 37 degrees Celsius overnight.



**Analysis:**
There was successful colony growth in the plate containing 50 uL of bacteria and SOC media. There were only three major colonies present on the plate containing 10 uL. It was expected that the 50 uL plate would contain more significant growth than the 10 uL plate. Colonies present likely contain transformed bacteria as they now produce a protein which confers resistance to the KAN which is present in the media. The KAN should have killed off any bacteria that did not incorporate the plasmid into their genome. This is not yet confirmed through more advanced analysis. The next step to confirm transformation is to over-express the protein in liquid media from colonies present.