Brianna+G.+(RP+Summ+14)

__ WEEK 9 (7/28/2014 - 8/1/2014) __

DNA samples from LIC-E master plate - B bacteria were sent to DNA sequencing. The DNA sequencing results were then BlastN-ed to the kpblaGES5 FASTA. (*Note: kpblaGES5 gene has 864 base pairs). Both tubes 8 and 9 had coverage from 825bp to 864bp.
 * DNA Sequenced DNA from mini prepped LIC-E Master Plate - B bacteria (7/29/2014) **

**Figure 1.** Tube 8 results blastN-ed to kpblaGES5 FASTA, where coverge was from 825bp to 864bp. This serves as an example of the average coverage the mini prepped DNA had with kpblaGES5.

The blastN results were similar to the DNA sequencing of the DNA from LIC-E master plate - A. They suggested that only a portion of the kpblaGES5 gene were being inserted into the BsaI digested pNIC. This may have occurred because there were floating segments of uncompleted kpblaGES5 gene in the gene sample used for cloning (it somehow surpassed the PCR clean up). This is weird since PCR clean up takes out DNA under 100bp and the recurring covered segment is 28bp long (should have been removed during the clean up). Another theory is that the rest of the gene was broken off after the back sticky end latched on to pNIC. This is also hard to believe because the gene hould have already been double stranded and bonded. The only way this could have occurred is if the hydrogen bonding between the DNA strands unbonded and the sense strand attached to the other end of the digested pNIC. --- NO CLONE PRODUCED

Spun down expression tubes 1-9 at 6000 rpm for 15min and expelled supernatant (pellet left). Tubes 1-7 had no pellet (discarded). Extracted DNA (potential clones) from bacteria (pellets in tubes 8 and 9) into 50uL of 5mM of Tris HCl.
 * Mini Prep of Expression Tubes from LIC-E (8/1/2014) **

__ Nanodrop concentration results of mini prepped bacteria: __ Tube 8: 37.35 ng/uL Tube 9: 29.75 ng/uL

Prepared a master plate of LIC-E plates A-C (plate D had no colonies). Made 9 expression tubes (5mL LB + 5uL kanamycin + bacteria from master plate).
 * Master Plate - B of LIC-E (7/31/2014) **


 * Figure1. ** Master plate - B of LIC-E plates A-C. Bacteria in zones 1-3 are from individual colonies in plate A. Bacteria in zone 4-6 are from individual colonies in plate B. Bacteria in zones 7-9 are from individual colonies in plate C. Photo taken after 16hr incubation period (8/1/14).

Digested mini prepped DNA from LIC-E master plate - A (all except DNA from tube 4 because of low concentration/not enough DNA) with SmaI restriction enzyme and visualized fragments on gel. In running gel, accidentally switched wires when bands were at the middle of the gel (bands traveled in opposite direction/ backwards for 15-20 minutes). Needed to run forward again (switched back wires) for 20 min. Gel showed that the mini prepped DNA from LIC-E master plate - A was not the expected clone of pNIC + GES5.
 * Restriction Enzyme Digest of LIC-E Master Plate - A DNA (7/30/2014)**


 * Figure 1. ** pNIC digested by smaI restriction enzyme.


 * Figure 2.** pNIC+GES5 (expected clone) digested by smaI restriction enzyme.


 * Figure 3.** How gel should look like of smaI digested pNIC+GES5, smaI digested pNIC, Full pNIC, and BsaI digested pNIC (larger portion of vector that was supposed to be used to transfer the kpblaGES5 gene into DH5alpha bacteria).


 * Figure 4.** Agarose gel of restriction enzyme smaI digest results of LIC-E master plate - A DNA. Lane 1 has 1kb ladder. Lane 2 has full pNIC. Lane 3 has pNIC digested by smaI. Lanes 4-7 has LIC-E master plate DNA from tubes 1-3 and 5 respectively, all digested by smaI.


 * Figure 4.** Agarose gel of restriction enzyme smaI digest results of LIC-E master plate - A DNA. Lane 1 has 1kb ladder. Lane 2 has full pNIC. Lane 3 has pNIC digested by smaI. Lanes 4-7 has LIC-E master plate DNA from tubes 6-9 respectively, all digested by smaI.

DNA samples from LIC-E master plate - A bacteria were sent to DNA sequencing. The DNA sequencing results were then BlastN-ed to the kpblaGES5 FASTA. (*Note: kpblaGES5 gene has 864 base pairs). Tube 3, 6, 8, and 9 had no coverage/similarity to kpblaGES5. Tubes 1, 4, and 5 had coverage from 825bp to 864bp. Tube 2 had coverage from 799bp to 858bp. Tube 7 had coverage from 834bp to 864bp.
 * DNA Sequenced DNA from mini prepped LIC-E Master Plate - A bacteria (7/29/2014) **

**Figure 1.** Tube 5 results blastN-ed to kpblaGES5 FASTA, where coverge was from 825bp to 864bp. This serves as an example of the average coverage the mini prepped DNA had with kpblaGES5.

The blastN results suggested that only a portion of the kpblaGES5 gene were being inserted into the BsaI digested pNIC. This may have occurred because there were floating segments of uncompleted kpblaGES5 gene in the gene sample used for cloning (it somehow surpassed the PCR clean up). This is weird since PCR clean up takes out DNA under 100bp and the recurring covered segment is 28bp long (should have been removed during the clean up). Another theory is that the rest of the gene was broken off after the back sticky end latched on to pNIC. This is also hard to believe because the gene hould have already been double stranded and bonded. The only way this could have occurred is if the hydrogen bonding between the DNA strands unbonded and the sense strand attached to the other end of the digested pNIC. --- NO CLONE PRODUCED

Spun down expression tubes 1-9 at 6000 rpm for 15min and expelled supernatant (pellet left). Extracted DNA (potential clones) from bacteria (pellets) and eluted in 50uL of 5mM Tris HCl.
 * Mini Prepped LIC-E Master Plate - A (7/29/2014) **

__ Nanodrop concentration results of mini prepped bacteria: __ Tube 1: 67.50 ng/uL Tube 2: 80.75 ng/uL Tube 3: 82.00 ng/uL Tube 4: 51.05 ng/uL Tube 5: 88.45 ng/uL Tube 6: 73.80 ng/uL Tube 7: 80.10 ng/uL Tube 8: 78.70 ng/uL Tube 9: 63.80 ng/uL

Prepared a master plate of LIC-E plate E. Made 9 expression tubes (5mL LB + 5uL kanamycin + bacteria from master plate).
 * Master Plate - A of LIC-E plate E (7/28/2014) **


 * Figure1. ** Master plate of LIC-E plates. Bacteria in zones 1-9 are from individual colonies in plate E. Photo taken after 16.5 hr incubation period (7/29/14).

__ WEEK 8 (7/21/2014 - 7/25/2014) __

Inserted kpblaGES5 into digested pNIC. Inserted resulting DNA into DH5alpha bacteria and began expression.
 * Annealing and Transformation (7/24/2014) **

Tube A: 8uL GES5E2 + 4uL Dig pNICD1C+ 25uL DH5alpha (2:1 ratio) Tube B: 9uL GES5E2 + 3uL Dig pNICD1C + 25uL DH5alpha (3:1 ratio) Tube C: 8uL GES5E2 + 2uL Dig pNICD1C + 25uL DH5alpha (4:1 ratio) Tube D: 10uL GES5E2 + 2uL Dig pNICD1C + 25uL DH5alpha (5:1 ratio) Tube E: 5uL GES5E2 + 2uL Dig pNICD1C + 11uL DH5alpha (Fun plate because I had sample left over)


 * Figure 1.** LIC-E tube A plate after 1 day (7/25) of 37C incubation. All of the yellow specs on the plate are bacterial colonies.


 * Figure 2.** LIC-E tube B plate after 1 day (7/25) of 37C incubation. All of the yellow specs on the plate are bacterial colonies.


 * Figure 3.** LIC-E tube C plate after 1 day (7/25) of 37C incubation. All of the yellow specs on the plate are bacterial colonies.


 * Figure 4.** LIC-E tube D plate after 1 day (7/25) of 37C incubation. All of the yellow specs on the plate are bacterial colonies.


 * Figure 5.** LIC-E tube E plate after 1 day (7/25) of 37C incubation. All of the yellow specs on the plate are bacterial colonies.

Prepared kpblaGES5-E2 insert (2 tubes) + digested pNIC-D1(7uL) -C (4uL) with T4 polymerase to create "sticky ends." Parafilmed all tubes during 75C heat shock and spun down all tubes after heat shock (revision based on major evaporation experience in LIC-D).
 * Cohesive End Generation (7/24/2014)**

Amplified a sample of completed kpblaGES5 gene (5 rounds). PCR cleaned up with 30 uL of 10mM Tris HCl (into 5 tubes). The bands in figure 1, lanes 1-5, showed all 5 rounds of PCR squared - E to be in the correct location (900bp) and were very clean. Lane 1 band was slightly faint but nothing serious. THere was contamination below the 500bp marker but was hopefully removed in PCR clean up.
 * PCR Squared - E using Secondary PCR - C2 and PCR Clean Up (7/22/2014) **


 * Figure 1.** Agarose gel of PCR squared - E, all 5 rounds distributed across lanes 1-5. Lane 6 has 1kb DNA ladder. Lanes 7-9 has digested pNIC - D1, D2, and D3 respectively. Lanes 1-5 have bands around 900bp with some smears below the 500bp. Lanes 7-9 have bands around the 2000bp and 5000bp with some smears above the upper 5000bp bands.

__Nanodrop average concentrations for PCR squared - E:__ Tube 1: 75.75 ng/uL Tube 2: 73.75 ng/uL Tube 3: 96.40 ng/uL Tube 4: 94.05 ng/uL Tube 5: 73.20 ng/uL

__Yield:__ Tube 1: 1969.5 ng Tube 2: 1917.5 ng Tube 3: 2506.4 ng Tube 4: 2445.3 ng Tube 5: 1903.2 ng

Inserted kpblaGES5 into digested pNIC. Inserted resulting DNA into DH5alpha bacteria and began expression.
 * Annealing and Transformation (7/23/2014) **

Tube A: 4uL GES5E1 + 2uL Dig pNICD1 + 30uL DH5alpha (2:1 ratio) Tube B: 6uL GES5E1 + 2uL Dig pNICD1 + 25uL DH5alpha (3:1 ratio) Tube C: 8uL GES5E1 + 2uL Dig pNICD1+ 30uL DH5alpha (4:1 ratio) Tube D: 5uL GES5E1 + 1uL Dig pNICD1 + 25uL DH5alpha (5:1 ratio)

After 1 hour of 37C incubation, the tubes sat in room temperature for 10-30 min, waiting for plates. Used plates from 6/16/2014.


 * Figure 1.** LIC-D tube A plate after 2 days (7/25) of 37C incubation. All of the yellow specs on the plate are sucrose crystals, not bacteria. No bacterial growth was observed.


 * Figure 2.** LIC-D tube B plate after 2 days (7/25) of 37C incubation. All of the yellow specs on the plate are sucrose crystals, not bacteria. No bacterial growth was observed.


 * Figure 3.** LIC-D tube C plate after 2 days (7/25) of 37C incubation. All of the yellow specs on the plate are sucrose crystals, not bacteria. No bacterial growth was observed.


 * Figure 4.** LIC-D tube D plate after 2 days (7/25) of 37C incubation. All of the yellow specs on the plate are sucrose crystals, not bacteria. No bacterial growth was observed.

The sucrose crystals should not have affected bacterial growth significantly however better plates should be used next time. With a lot of evaporation during the 75C heat shock, much of the DNA (gene + vector) was probably lost and thus, no clones would have been able to enter and transform the DH5alpha bacteria -- explanation for why no bacterial growth appeared on the plates after 2 days.

Prepared kpblaGES5-E1 insert (2 tubes) + digested pNIC-D1 with T4 polymerase to create "sticky ends." T4 treated kpblaGES5-E tubes and pNIC tubes reduced drastically in volume after the 75C heat shock (about half lost due to extensive evaporation).
 * Cohesive End Generation (7/23/2014)**

Amplified a sample of completed kpblaGES5 gene (1 round). PCR cleaned up with 30 uL of 10mM Tris HCl. Made in case more gene sample was needed for a cloning procedure. Wited 5-10min for PCR machine to turn on. The bands in figure 1, lanes 2-5, showed no bands - no gene produced. This may be due to the 5-10min wait for the PCR machine in room temperature (denature DNA) but it is unlikely since DNA can withstand room temperature for a longer period of time than 5-10 min. It could also be that something was not added to the PCR squared mix by mistake.
 * PCR Squared - F using Secondary PCR - C3 and PCR Clean Up (7/22/2014) **


 * Figure 1.** Agarose gel of 1kb DNA ladder in lane 1 and PCR squared - F. No bands apparent - no kpblaGES5 gene produced.

Digested pNIC - D (6 rounds) with BsaI and PCR cleaned up with 30uL of 10mM Tris HCl (into 3 tube) from the PCR clean up kit. Bands in agarose gel (figure 1, lanes 7-9, in PCR Squared - E procedure 7/22/2014) were in the correct locations (5000bp and 2000bp). The lower bands were much cleaner than the upper bands which had streaks above it. Despite the streaks, the digested pNIC would still be used for cloning procedures.
 * Digested pNIC - D using pNIC Full - A and PCR Clean Up (7/21/2014) **

__Nanodrop average concentrations for Digested pNIC - D:__ Tube 1: 66.2 ng/uL Tube 2: 65.9 ng/uL Tube 3: 66.2 ng/uL

__Yield:__ Tube 1: 1396.5 ng Tube 2: 1383.9 ng Tube 3: 1390.0 ng

Amplified a sample of completed kpblaGES5 gene (5 rounds). The agarose gel of PCR squared - D had no bands, meaning that the procedure did not amplify the kpblaGES5 gene at all. This may have occurred because the Q5 polymerase used had been prepared (dilute to 1ug/uL) a week before and stored in the -20C freezer.
 * PCR Squared - D using Secondary PCR - C (7/21/2014) **


 * Figure 1. ** Agarose gel of a 1kb DNA ladder in lane 1 and all 5 PCR squared - D rounds distributed across lanes 2-6. No bands apparent in lanes 2-6.

__ WEEK 7 (7/14/2014 - 7/18/2014) __

Amplified a sample of completed kpblaGES5-B gene (1 round). PCR cleaned up with 30 uL of 10mM Tris HCl. Bands (figure 1, lanes 6-9) were found to be in correct location (around 900bp). Bands 6 and 7 were very faint and band 8 was less faint, more detectable. Band 9 on the other hand was much more distinct. It could be that the PCR squared - C did not work very well for those the DNA in tubes 6-8, however all would still be combined for PCR clean up. Streaks were idenfied under the 500bp marker for lanes 8 and 9 but it was hoped that PCR clean up would get rid of them. During the PCR clean up procedure, about 200uL of binding solution + PCR squared -gene combined was lost (pipetting error/ clumsy/ spill).
 * PCR Squared - C using Secondary PCR - C+ PCR Clean Up (7/18/2014)**


 * Figure 1. ** Agarose gel of secondary PCR-C with 4 rounds distributed across lanes 1-4. Lane 5 contained the 1lb DNA ladder. Lanes6-9 represented each tube from 1 round of PCR squared - C. Bands in lanes 1-4 and 6-9 were located around 900bp marker. Some faint streaks were identified in those lanes under the 500bp marker.

__Average kpblaGES5-B concentration:__ 45.9 ng/uL __Yield:__ 2111.4 ng

Because I used all of my kpblaGES5 sample in LIC-C, more kpblaGES5 was needed to do another cloning procedure. Amplified completed kpblaGES5 using primary PCR - A sample (4 rounds). Refer to figure 1, lanes 1-4, in PCR Squared - C (7/18/2014) procedure for gel. Bands were good, all around 900bp (correct) location. Very faint streaks were identified above the bands but they do not seem like serious contamination. There were smudges however below the 500bp but those smudges would hopefully be removed from the sample during PCR clean-up, after PCR square - C.
 * Secondary PCR - C using Primary PCR - A (7/18/2014) **

Inserted kpblaGES5 into digested pNIC. Inserted resulting DNA into DH5alpha bacteria and began expression.
 * Annealing and Transformation - C (7/17/2014) **

Tube A: 4uL GES5A + 2uL Dig pNICC + 25uL DH5alpha (2:1 ratio) Tube B: 12uL GES5A + 4uL Dig pNICC + 25uL DH5alpha (3:1 ratio) Tube C: 9.5uL GES5B + 5uL Dig pNICB + 25uL DH5alpha (2:1 ratio) Tube D: 9.5uL GES5B + 2uL Dig pNICB + 25uL DH5alpha (5:1 ratio) Tube E: 8uL GES5B + 2uL Dig pNICB + 25uL DH5alpha (4:1 ratio) Tube F: 6uL GES5B + 3uL Dig pNICB + 25L DH5alpha (2:1 ratio)

Used fresh plates made 10 min before and because the plates were placed too close to the bunsen burner, the LB agar melted with bacteria sample on it. All the plates ended up having indents because of the collirollers on it while melting. Lost plate with tube F sample because it was super liquidy, took out the collirollers by hand (probably super contaminated), and the plate never solidified. All plate were expected to fail because of the LB agar condition.

Plates were found to be super contaminated on 7/18/2014. Plates were either made super contaminated and/or the incubator was contaminated and needed cleaning with ethanol and/or 10% bleach. Next time, clean, incubator with ethanol and 10% bleach and make own plates that are super solidified before used.


 * Figure 1.** LIC-C tube A plate after 1 day (7/18) of 37C incubation. All of the yellow specs/smudges are contaminated/unwanted bacteria.


 * Figure 2.** LIC-C tube B plate after 1 day (7/18) of 37C incubation. All of the yellow specs/smudges are contaminated/unwanted bacteria.


 * Figure 3.** LIC-C tube C plate after 1 day (7/18) of 37C incubation. All of the yellow specs/smudges are contaminated/unwanted bacteria.


 * Figure 4.** LIC-C tube D plate after 1 day (7/18) of 37C incubation. All of the yellow specs/smudges are contaminated/unwanted bacteria.


 * Figure 5.** LIC-C tube E plate after 1 day (7/18) of 37C incubation. All of the yellow specs/smudges are contaminated/unwanted bacteria.

Prepared kpblaGES5-A and -B insert + digested pNIC-B and -C with T4 polymerase to create "sticky ends." All went 10min over the room temperature incubation.
 * Cohesive End Generation - C (7/17/2014) **

Spun down expression tubes 1-9 at 6000 rpm for 20min and expelled supernatant (pellet left). Tube 9 had no pellet (discarded). Extracted DNA (potential clones) from bacteria (pellets).
 * Mini Prep of Expression Tubes from LIC-B (7/18/2014) **

__ Nanodrop concentration results of mini prepped bacteria: __ Tube 1: 11.1 ng/uL Tube 2: 8.8 ng/uL Tube 3: 5.1 ng/uL Tube 4: 4.4 ng/uL Tube 5: 4.8 ng/uL Tube 6: 5.3 ng/uL Tube 7: 11.3 ng/uL Tube 8: 6.5 ng/uL

The concentrations of the mini prepped DNA from LIC-B were way too low (probably just gathered random DNA/contamination or probably nothing gathered at all). This was indicative of useless DNA - not the clone of pNIC + kpblaGES5. All mini prepped samples were discarded.

Prepared a master plate of LIC-B plates A-C. Made 9 expression tubes (5mL LB + 5uL kanamycin + bacteria from master plate).
 * Master Plate of LIC-B (7/17/2014) **


 * Figure1. ** Master plate of LIC-B plates A-C. Bacteria in zones 1-3 are from individual colonies in plate A. Bacteria in zone 4-6 are from individual colonies in plate B. Bacteria in zones 7-9 are from individual colonies in plate C. Photo taken after 16hr incubation period (7/18/14).

After taking master plate of LIC-B out of incubator, plate was tilted. Bacterial colonies were liquidy and ran off to other zones. Master plate was watery probably because plate was made incorrectly.

Amplified a sample of completed kpblaGES5-B gene. Dyed 1 tube sample on accident and lost about 10uL of sample. PCR cleaned up with 30 uL of 10mM Tris HCl. Bands (figure 1, lanes 2-5) were good in that they were distinct and in correct location (around 900bp). The bands were faint, maybe because only 5uL of sample was used for the gel or hardly any DNA was made/amplified in PCR squared procedure. In addition, streaks were found below all the sample bands (below the 500bp marker). It was hopped that they could be easily taken out during PCR clean up.
 * PCR Squared - B using Secondary PCR - B + PCR Clean Up (7/16/2014) **


 * Figure 1. ** Agarose gel of 1kb ladder in lane 1 and PCR squared of kpblaGES5 - B in lanes 2-5. In lanes 2-5, bands are found around 900bp and streaks below 500bp.

__Average kpblaGES5-B concentration:__ 70.1 ng/uL __Yield:__ 1682.4 ng

Digested pNIC - B (4rounds) with BsaI and PCR cleaned up with 30uL of 10mM Tris HCl (into 1 tube) from the PCR clean up kit. Bands in agarose gel (figure 1, lane 2) came out at correct locations (2000bp and 5000bp) compared to 1kb ladder however the upper band appeared as if it were a cluster of multiple bands nad has a streak above it. Upper streak w probably colied up DNA as in digested pNIC - B. pNIC would still be used for cloning procedure.
 * Digested pNIC - C + PCR Clean up (7/16/2014) **


 * Figure ** **1.** Agarose gel of 1kb DNA ladder and digested pNIC-C in lane 2 (bands around 2000bp and 5000bp).

__Average Digested pNIC - B concentration:__ 120.55 ng/uL __Yield:__ 2531.55 ng

Inserted kpblaGES5 into digested pNIC. Inserted resulting DNA into DH5alpha bacteria and began expression.
 * Annealing and Transformation - B (7/15/2014) **

Tube A: 6uL GES5A + 10uL Dig pNICA + 10uL DH5alpha (3:5 ratio) Tube B: 5uL GES5A + 10uL Dig pNICA + 20uL DH5alpha (1:2 ratio) Tube C: 8uL GES5A + 4uL Dig pNICA + 20uL DH5alpha (2:1 ratio)


 * Figure 1.** LIC-B tube A plate after 2 days (7/17) of 37C incubation. 4 colonies identified.


 * Figure 2.** LIC-B tube B plate after 2 days (7/17) of 37C incubation. 5 colonies identified.


 * Figure 3.** LIC-B tube C plate after 2 days (7/17) of 37C incubation. 3 colonies identified.

Prepared kpblaGES5-A insert and digested pNIC-A with T4 polymerase to create "sticky ends."
 * Cohesive End Generation - B (7/15/2014) **

Digested pNIC - B with BsaI and PCR cleaned up with 30uL of 10mM Tris HCl from the PCR clean up kit. Made as back-up in case another cloning procedure was conducted. Bands in agarose gel (figure 1, lane 7) came out very nice - very clean and defined at correct locations compared to 1kb ladder.
 * Digested pNIC - B + PCR Clean-up(7/14/14) **


 * Figure ** **1.** Agarose gel of 1kb DNA ladder, ignore lanes 2-5 (not my samples), secondary PCR - B in lane 2 (band around 900bp), and digested pNIC-B in lane 3 (bands around 2000bp and 5000bp).

__Average Digested pNIC - B concentration:__ 56.5 ng/uL __Yield:__ 791 ng

Inserted kpblaGES5 into digested pNIC. Inserted resulting DNA into DH5alpha bacteria and began expression.
 * Annealing and Transformation - A of CEG-A(7/14/2014) **

Tube A: 4uL GES5A + 2uL Dig pNICA + 5uL DH5alpha (DNA sample splattered on sides of tube, hard to tell if vector and gene made contact) Tube B: 1uL GES5A + 5uL Dig pNICA + 25uL DH5alpha Tube A': 4uL GES5A + 2uL Dig pNICA + 25uL DH5alpha (to make up for tube A mess up)

Accidentally used plates from 7/16/13 (over a year old - expired).


 * Figure 1.** LIC-A tube A plate after 2 days of 37C incubation. No growth visible.


 * Figure 2.** LIC-A tube B plate after 2 days of 37C incubation. No growth visible.


 * Figure 3.** LIC-A tube A' plate after 2 days of 37C incubation. No growth visible.

No growth visible after 2 days, probably due to: expired plate, longer heat shock, gene and vector could not connect or make contact, engineered clone could not enter bacteria.

Prepared kpblaGES5-A insert and digested pNIC-A with T4 polymerase to create "sticky ends."
 * Cohesive End Generation-A (CEG-A) of kpblaGES5-A and digested pNIC28-Bsa4-A (7/14/2014) **

__ WEEK 6 (7/7/2014 - 7/11/2014) __

Amplified a sample of completed kpblaGES5-A gene, to compensate for the Secondary PCR - A that went missing. Band on gel came out well, slightly smudgy, around the 900bp, which is still good (supposed to be around 864bp).
 * Secondary PCR - B (7/11/2014) **

--> for agarose gel of band, refer to lane 6 in figure 1 of the Digested pNIC - B (7/14/14) procedure in week 7.

Digested pNIC28-Bsa4-A with BsaI and PCR cleaned up with 30uL of Elution Solution from the PCR clean up kit.
 * Digested pNIC28-Bsa4-A and PCR cleaned-up (7/11/2014) **




 * Figure** **1.** Nanodrop result of digested pNIC-A run 1, shown as 10mm absorbance vs wavelength nm. The concentration measured was 34.2 ng/uL.


 * Figure 2.** Nanodrop result of digested pNIC-A run2, shown as 10mm absorbance vs wavelength nm. The concentration measured was 33.4ng/uL.

Average digested pNIC-A concentration: 33.8ng/uL Yield after nanodropping: 878.8ng

Low concentration and low yield. Would still be used for ligation independent cloning.

Sent in pNIC28-Bsa4 for DNA sequencing, one with primer pLIC-for and the other with primer pLIC-rev.
 * DNA sequencing of pNIC28-Bsa4 (7/10/2014) **

code >xbgPNICfor-pLIC-for 1267 31 765 0.05 NNNNNNNNNNNNTNNACTTTAGNNGAGANNTACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGGAGACCGACGTCCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATAAAGTTCCTGAGTTCGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGNCTGGACGTTTGGGACAGCTGGCCATTACAAAACACTGACGGCACTGTCGCAAACTATCACGGCTNCCNNNNNTCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGNNNAAACTTCTATTGACAGCTGGAAAAACGCTGNCNGCGTCNTTAANNCAGCGANAANTCNATGCAAATGATTCTATCCTAAANACCAAACANANANNGNCNNNNANCNCNTTTNCNTCTGANGNAAATCNNNNTCTANNCTGATTNNNNNAANNNTNNNNNAANAANNNCTGNNNNTNNNNNNNNTNNNNNNCNNCNTNNNNNNNNNNNNNNNNNNNNNNNGGNNNANNNNNNNANNNNNNNNNNNNNNGNNNNNNANNNNNNNNNNNNNGNNNNNNNNNNNNNNNGNNNNNNNNNNNNNNGNNNNNNNNNNNNNNN code

code >xbgPNICrev-pLIC-rev 1275 30 805 0.05 NNNNNNNNNNNNNNNNNCTCNGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGATCCGTATCCACCTTTACTGGAGACCGTCAATGCCAATAGGATATCGGCATTTTCTTTTGCGTTTTTATTTGTTAACTGTTAATTGTCCTTGTTCAAGGATGCTGTCTTTGACAACAGATGTTTTCTTGCCTTTGATGTTCAGCAGGAAGCTAGGCGCAAACGTTGATTGTTTGTCTGCGTAGAATCCTCTGTTTGTCATATAGCTTGTAATCACGACATTGTTTCCTTTCGCTTGAGGTACAGCGAAGTGTGAGTAAGTAAAGGTTACATCGTTAGGATCAAGATCCATTTTTAACACAAGGCCAGTTTTGTTCAGCGGCTTGTATGGGCCAGTTAAAGAATTAGAAACATAACCAAGCATGTAAATATCGTTAGACGTAATGCCGTCAATCGTCATTTTTGATCCGCGGGAGTCAGTGAACAGGTACCATTTGCCGTTCATTTTAAAGACGTTCGCGCGTTCAATTTCATCTGTTACTGTGTTAGATGCAATCAGCGGTTTCATCACTTTTTTCAGTGTGTAATCATCGTTTAGCTCAATCATACCGAGAGCGCCGTTTGCTAACTCAGCCGTGCGTTTTTTATCGCTTTGCAGAAGTTTTTGACTTTCTTGACGGAAGAATGATGTGCTTTTGCCATAGTATGCTTTGTTAAATAAAGATTCTTCGCCTTGGTAGCCATCTTCAGTTCCAGTGTTTGCTTCAAATACTAAGTATTTGTGGCCTTTATCTTCTACGTAGTGAGGATCTCTCAGCGTATGGNTGTCGCCTGAGCTGTAGTTGCCTTCATCGATGAACTGCTGTACATTTTGATACGTTTTTCCGTCACCGTCAAAGATTGATTTATAATCCTCTACACCGNTGATGTTCAAAGAGCTGTCTGATGCTGATACGTTAACTTGTGCAGTTGTCAGTGNTTGNTTGNCGTAATGNTTANCNGANAAATCAGTGTAGANTAAACNNANTTTTNCGTCNGANGTAAATGNGNTGANNTGACNTTNNNNNGNTNNGNCNTTTAGNNNAGNATCNTTNCATCNNNNNNCGCNNNNNTNNNNANNNNNNNCNNNTNNNNCNNNCAATNNNNNTTNNNCNANTTTTNNNNNNNANNNNNNNNNCNNNNNNNNNTNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNGNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNNN code

BlastN results of pNIC-for results to PNIC28-Bsa4 CDS

BlastN result of pNIC-rev results to pNIC28-Bsa4 CDS

The DNA sequencing results of the midi prepped pNIC28-Bsa4 has an average 98% identity to the known CDS, which is really good but a total query cover of 27% which is what pLIC-for and pLIC-rev were supposed to cover. The midi prepped pNIC28-Bsa4 is now verified and can be used for digesting and ligation independent cloning with kpblaGES5 insert.

Spun down LB with transformed DH5alpha with pNIC28-Bsa4 at 6000xg for 15 min. Extracted pNIC28-Bsa4 from DH5alpha bacteria and purified via midi prep.
 * Midi prepped pNIC28-Bsa4 **** (7/9/2014) **

Weights of pellets: Tube A: 0.15g Tube B: 0.23g Tube C: 0.17g Tube D: 0.16g Tube E: 0.18g Tube F: 0.27g Tube G: 0.21g Tube H: 0.10g


 * Figure 1.** Nanodrop result of pNIC28-Bsa4 after midi prep, run 1, shown as 10mm absorbance vs wavelength nm. The concentration measured was 102.7ng/uL.


 * Figure 2.** Nanodrop result of pNIC28-Bsa4 after midi prep, run 2, shown as 10mm absorbance vs wavelength nm. The concentration measured was 108.4ng/uL.

Average full pNIC28-Bsa4 concentration: 105.55ng/uL Yield after nanodrop, gel, and DNA sequencing: 52775ng


 * Figure 3.** Agarose gel of 1kb ladder in lane 1 and pNIC28-Bsa4 in lane 2.

The gel should have run for 5 more minutes to spread out the ladder but the resutls are still observable. pNIC28-Bsa4 appeared in the correct location (pNIC-28-Bsa4 has 7284bp and landed between the 6000 and 8000bp ladder markers). there is however a lighter band above the 7284bp band. This is probably some of the pNIC-Bsa4 coiled and did not travel correctly because of it (common for large plasmids). pNIC28--Bsa4 location is good and could be used for digestion and ligation independent cloning with kpblaGES5 insert.

Digested pGBR22 plasmid with restriction enzymes EcoRI (in NEBuffer EcoRI), PvuII (NEBuffer 2), and both EcoRI and PvuII (NEBuffer 2) and visualized fragments on gel.
 * Restriction Enzyme Digest of pGBR22 with EcoRI and PvuII **** (7/9/2014) **


 * Figure 1. ** Agarose gel of digested pgbr22. Lane 1 had the 1 kb ladder. Lane 2 has an uncut pgbr22. Lane 3 has pgbr22 digested by EcoRI. Lane 4 has pgbr22 digested by PvuII. Lan 5 has pgbr22 digested by EcoRI and PvuII.

Overall, the bands appeared where they were supposed to according to the virtual gel. Lane 3 however had multiple bands that may just be because of the coiling of the DNA and the resulting effect of staggered traveling. Lane 4 and 5 also appeared to have contamination but that may just also be staggered based on the coiling of the DNA.

Took some transformed DH5alpha with pNIC28-Bsa from a plate (made by Zain 6/14/2014) and expressed in 160mL of LB media overnight (16hr 37C incubation).
 * Expressing pNIC28-Bsa4 in transformed DH5alpha bacteria (7/8/2014) **

Purified kpblaGES5 after PCR Squared.
 * PCR Cleanup of kpblaGES5 (7/8/2014) **


 * Figure 1.** Nanodrop result of kpblaGES5-A after PCR clean-up, run 1, shown as 10mm absorbance vs wavelength nm. The concentration measured was 177.1ng/uL.


 * Figure 2.** Nanodrop result of kpblaGES5_A after PCR clean-up, run 2, shown as 10mm absorbance vs wavelength nm, The concentration measured was 174.0 ng/uL.

Average kpblaGES5 concentration: 175.55ng/uL Yield after nanodropping: 7174.72ng

Amplified complete kpblaGES5 to a large extent to make sure enough kpblaGES5 is available for cloning (insertion into vector pNIC28-Bsa4).
 * PCR Squared of kpblaGES5 (7/8/2014) **


 * Figure 1.** Agarose gel of 1kb ladder in lane 1. PCR squared of kpblaGES5 in lanes 5-8.

PCR squared results appeared to be in the correct location (around 864bp). There are large smears for each PCR squared lanes (above and below bands). This might be an indication of contamination. The below smears might be some uncompleted kpblaGES5 from PCR but may be removed in PCR clean up.

__ WEEK 5 (6/30/2014 - 7/3/2014) __

Assembled kpblaGES5 from primary PCR was amplified to a moderate extent to make sure the complete kpblaGES5 amplified correctly. An agarose gel was then ran to verify the product.
 * Secondary PCR of kpblaGES5 (7/3/2014) **


 * Figure 1. ** Agarose gel of 1kb ladder in lane 3, primary PCR of kpblaGES5 in lane 1 and secondary PCR kpblaGES5 in lane 2.

The smear is perfect for lane 1 (primary PCR). There was a small bit of contamination in lane 2 (secondary PCR) but the band is around 900bp (kpblaGES5 is 864bp - correct band location). The secondary PCR product is good to go for PCR squared.

Diluted the primers designed in the " PCR Primer Design Tails for pNIC-Bsa4 Cloning lab (6/30/2014)." 263uL of TE was added to the 0.0263umol kpGES-for and 251uL of TE was added to the 0.0251umol kpGES-rev, to make both of them 100uM stock solutions. 200uL of 20uM dilution of each stock primer was then made in separate 1.7mL centrifuge tube.
 * Primer Dilution of kpGES5-for and kpGES5-rev (7/3/2014) **

Secondary polishing step to purify YopH from others in the Ni-NTA purified sample, based on differences in size.
 * FPLC Gel Filtration of YopH (7/2/2014) **


 * Figure 1.** FPLC result of YopH. X-axis in black was the amount of YopH elution was released. The x-axis red markers were the tube numbers of the FPLC machine. The y-axis blue is the UV absorbance/concentration of the elution sample. The dotted line is the protein ladder based on weight (kDa) and the solid blue line is the protein measured from the sample. The first peak of the dotted blue line marks 75kDa. The second peak of the dotted blue line marks 44kDa. The third peak of the dotted blue line marks 33kDa. The first and second peak of the solid blue line indicates that protein was identified and put into tubes corresponding to the red tick marks.

Tubes 29-37 were collected for 1 protein sample and tubes 39-46 were collected for the other protein. The weight of YopH is 36kDa so the second collection (39-46) is probably YopH, considering it is between the 44kDa and 33kDa markers. The first (29-37) tubes is probably another protein since it is about 44kDa. It is probably part of the BL21(DE3) bacteria and has a his-tag like YopH. The normal BL21(DE3) genome and the pNIC28-Bsa4 vector with YopH insert would have to be translated and studied to see if a protein could be produced with those features and be identified as to what it is. The second collection however can be used for next enzyme assays to test YopH functionality.

Assembled the oligo set, filling the gaps between the oligos and piecing together a full length kpblaGES5. (agarose gel of primary PCR kpblaGES5 product in figure 1, lane 2 in the secondary PCR of kpblaGES5 report 7/3/2014)
 * Primary PCR of kpblaGES5 (7/1/2014) **

Made a single mix of the segments of the kpblaGES5 gene ordered from IDT. These segments would be used in PCR for assembly and amplification.
 * Made Oligo Mix of kpblaGES5 (7/1/2014) **

Amplified pNIC28-Bsa4 vector using pLIC-for forward primer and pLIC-rev reverse primer. Tube A had 0.0463ng of pNIC-Bsa4, tube B had 0.463ng of pNIC-Bsa4, tube C had 4.63ng of pNIC-Bsa4, and tube D had no DNA thus serving as a control. Replicated pNIC-Bsa4 was verified by an agarose gel.
 * PCR of pNIC-Bsa4 using pLIC primers (6/30/2014) **


 * Figure 1. ** PCR agarose gel for pNIC28-Bsa4. Lane 1 contains the 100bp DNA ladder and lane 10 contains a 1 kb ladder from New England Biolabs. Lanes 2-5 contain PCR DNA samples A-D respectively for this lab (Brianna). Lanes 6-9 contain PCR DNA samples for Luis. The absence of sample in lane 7 is due to a hole in the well through which all of the sample fell through when being loaded.


 * Lane 1: ** 100 bp DNA Ladder
 * Lane 2: ** 0.0463 ng DNA template (Brianna)
 * Lane 3: ** 0.463 ng DNA template (Brianna)
 * Lane 4: ** 4.63 ng DNA template (Brianna)
 * Lane 5: ** Control, No DNA template (Brianna)
 * Lane 6: ** 0.0463 ng DNA template (Luis)
 * Lane 7: ** 0.463 ng DNA template (Luis)
 * Lane 8: ** 4.63 ng DNA template (Luis)
 * Lane 9: ** Control, No DNA template (Luis)
 * Lane 10: ** 1kb DNA Ladder

pNIC-Bsa4 should have been around the 8000bp mark since it is 7184bp long; however, 2 bands appeared, one at the 200bp mark and the other at the 1000bp. This may have been because the PCR process was incomplete. The top band did get darker as more DNA was added (lanes 2 to 4) which is what was expected. More DNA enables more dye to bind. There was a random mark on the control (No DNA) which implies that there was contamination across the board (lanes 2-5). This may have affected the incompletion of the pNIC28-Bsa4 PCR.

Designed a forward and reverse primer for PCR amplifying the CDS of kpblaGES5 (target gene for ges-5 carbapenemase) sequence and synthesized compatible ends suitable for ligation independent cloning for insertion into the pNIC-Bsa4 as the accepting vector for eventual expression.
 * PCR Primer Design Tails for pNIC-Bsa4 Cloning (6/30/2014) **

__ Forward Primer: __ 5’ TACTTCCAATCCATG __CGTTTCATCCACG __ 3’ 28 bp GC Content 46.4 % 0 mM Mg2+ **Tm 61.3 oC** 1.5 mM Mg2+ **Tm 68.2 oC** 2 mM Mg2+ **Tm 68.8 oC** 4 mM Mg2+ **Tm 69.9 oC** 6 mM Mg2+ **Tm 70.4 oC**

__ Reverse Primer __ : 5’ TATCCACCTTTACTGTTA __TTTGTCGGTAGACAGG __ 3’ 34 bp GC Content 41.2 % 0 mM Mg2+ **Tm****60.7 oC** 1.5 mM Mg2+ **Tm 68.2 oC** 2 mM Mg2+ **Tm 68.7 oC** 4 mM Mg2+ **Tm****69.7 oC** 6 mM Mg2+ **Tm 70.1 oC**

TACTTCCAATCC ATG __CGTTTCATCCACG __CGCTCCTGCTGGCTGGTATCGCGCACTCTGCGTATGCGTCTGAAAAACTCACCTTTAAAACCGA CCTGGAAAAGCTCGAACGCGAGAAGGCCGCTCAAATCGGCGTGGCGATCGTTGACCCACAGGGTGAAATTGTAGCGGGTCACCGTATGGCTCA ACGCTTCGCGATGTGCTCTACCTTCAAATTCCCACTGGCTGCGCTCGTTTTTGAACGTATCGACTCTGGTACTGAGCGTGGTGACCGTAAGCTCT CCTACGGTCCGGATATGATTGTTGAATGGTCTCCGGCGACCGAACGTTTCCTGGCGTCTGGTCACATGACTGTACTGGAAGCGGCGCAGGCAG CCGTTCAGCTGTCTGATAACGGTGCGACCAACCTGCTGCTGCGCGAAATCGGTGGTCCAGCGGCCATGACCCAGTATTTCCGTAAGATCGGTGA CTCTGTTTCTCGTCTGGACCGCAAGGAACCGGAAATGTCTGACAATACCCCGGGTGACCTCCGTGACACGACGACCCCGATCGCAATGGCGCG TACCGTTGCGAAAGTTCTGTACGGTGGTGCGCTGACGTCCACCTCTACCCACACCATCGAGCGCTGGCTCATCGGTAACCAGACTGGTGATGCG ACGCTGCGTGCGGGTTTCCCGAAAGACTGGGTTGTTGGCGAAAAGACCGGCACCTGTGCGAATGGTGGTCGTAACGACATCGGTTTCTTCAAA GCGCAGGAACGTGACTACGCGGTTGCTGTTTACACTACTGCGCCTAAACTGTCTGCGGTTGAGCGCGATGAACTGGTTGCGTCCGTTGGTCAG GTTATCACCCAACTCATCCTGTCTACCGACAAA TAA CAGTAAAGGTGGATA
 * Insert: blaGES5 FASTA sequence**

TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGA GATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATG GAGACCGACGTCCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTA AAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTT CTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAA CGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGT GTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCAT CTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGAACATCAA AAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTTGCGA AAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAATCCCT GAACAGCAAAAAAATGAAAAATATAAAGTTCCTGAGTTCGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGC CTGGACGTTTGGGACAGCTGGCCATTACAAAACACTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCT TTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTA TTGACAGCTGGAAAAACGCTGGCCGCGTCTTTAAAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAGAC CAAACACAAGAATGGTCAGGTTCAGCCACATTTACATCTGACGGAAAAATCCGTTTATTCTACACTGATTTCTCCGGT AAACATTACGGCAAACAAACACTGACAACTGCACAAGTTAACGTATCAGCATCAGACAGCTCTTTGAACATCAACGG TGTAGAGGATTATAAATCAATCTTTGACGGTGACGGAAAAACGTATCAAAATGTACAGCAGTTCATCGATGAAGGCAA CTACAGCTCAGGCGACAACCATACGCTGAGAGATCCTCACTACGTAGAAGATAAAGGCCACAAATACTTAGTATTTG AAGCAAACACTGGAACTGAAGATGGCTACCAAGGCGAAGAATCTTTATTTAACAAAGCATACTATGGCAAAAGCACA TCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAAAGCGATAAAAAACGCACGGCTGAGTTAGCAAACGGCGCTCT CGGTATGATTGAGCTAAACGATGATTACACACTGAAAAAAGTGATGAAACCGCTGATTGCATCTAACACAGTAACAG ATGAAATTGAACGCGCGAACGTCTTTAAAATGAACGGCAAATGGTACCTGTTCACTGACTCCCGCGGATCAAAAATG ACGATTGACGGCATTACGTCTAACGATATTTACATGCTTGGTTATGTTTCTAATTCTTTAACTGGCCCATACAAGCCGC TGAACAAAACTGGCCTTGTGTTAAAAATGGATCTTGATCCTAACGATGTAACCTTTACTTACTCACACTTCGCTGTACC TCAAGCGAAAGGAAACAATGTCGTGATTACAAGCTATATGACAAACAGAGGATTCTACGCAGACAAACAATCAACGT TTGCGCCTAGCTTCCTGCTGAACATCAAAGGCAAGAAAACATCTGTTGTCAAAGACAGCATCCTTGAACAAGGACAA TTAACAGTTAACAAATAAAAACGCAAAAGAAAATGCCGATATCCTATTGGCATTGACGGTCTCCAGTAAAGGTGGATA CGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCG GCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGG CCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTA GCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCC GCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCT TTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCA TCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAA CAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAG CTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGT GCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCA TCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGA AGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACA TCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCC GGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCA CTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACA ATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGA TATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATA AAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCA ACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGA TTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCA AGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGAC CAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATC CTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAG AGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCG TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGC TGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGC TGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAG CTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGG AGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTT GAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTT CCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCC TTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGA GCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATC TGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACA CCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGT CTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCA GCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAA TGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGG ATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATG CCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGG TCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGG AACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGT TGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAG TAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATG CCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGC AAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACC CAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGC CCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAG TGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAAT GAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGG GCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCA GGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACC GAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCA ACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTC GCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCC GAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGT CGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAAC ATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTT GCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGG CACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGG CAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCAT CGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAA GAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGG GCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGAC TCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAG GAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCC CGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGG TGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAAT
 * Accepting Vector: pNIC28-Bsa4 FASTA sequence **

TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGA GATATACATATG CACCATCATCATCATCAT TCTTCTGGTGTAGATCTGGGTACCGAGA ACCTGTACTTCCAAT__CC__ __ ATG C __ __<span style="font-family: Calibri,sans-serif;">GTTTCATCCACGCGCTCCTGCTGGCTGGTATCGCGCACTCTGCGTATGCGTCTGAAAAACTCACCTTTAAAACCGACCTGGAAAAGCTCGAACG __ __<span style="font-family: Calibri,sans-serif;">CGAGAAGGCCGCTCAAATCGGCGTGGCGATCGTTGACCCACAGGGTGAAATTGTAGCGGGTCACCGTATGGCTCAACGCTTCGCGATGTGCTC __ __<span style="font-family: Calibri,sans-serif;">TACCTTCAAATTCCCACTGGCTGCGCTCGTTTTTGAACGTATCGACTCTGGTACTGAGCGTGGTGACCGTAAGCTCTCCTACGGTCCGGATATGA __ __<span style="font-family: Calibri,sans-serif;">TTGTTGAATGGTCTCCGGCGACCGAACGTTTCCTGGCGTCTGGTCACATGACTGTACTGGAAGCGGCGCAGGCAGCCGTTCAGCTGTCTGATAA __ __<span style="font-family: Calibri,sans-serif;">CGGTGCGACCAACCTGCTGCTGCGCGAAATCGGTGGTCCAGCGGCCATGACCCAGTATTTCCGTAAGATCGGTGACTCTGTTTCTCGTCTGGAC __ __<span style="font-family: Calibri,sans-serif;">CGCAAGGAACCGGAAATGTCTGACAATACCCCGGGTGACCTCCGTGACACGACGACCCCGATCGCAATGGCGCGTACCGTTGCGAAAGTTCTG __ __<span style="font-family: Calibri,sans-serif;">TACGGTGGTGCGCTGACGTCCACCTCTACCCACACCATCGAGCGCTGGCTCATCGGTAACCAGACTGGTGATGCGACGCTGCGTGCGGGTTTC __ __<span style="font-family: Calibri,sans-serif;">CCGAAAGACTGGGTTGTTGGCGAAAAGACCGGCACCTGTGCGAATGGTGGTCGTAACGACATCGGTTTCTTCAAAGCGCAGGAACGTGACTAC __ __<span style="font-family: Calibri,sans-serif;">GCGGTTGCTGTTTACACTACTGCGCCTAAACTGTCTGCGGTTGAGCGCGATGAACTGGTTGCGTCCGTTGGTCAGGTTATCACCCAACTCATCCT __ __<span style="font-family: Calibri,sans-serif;">GTCTACCGACAAA __ TAA CAGTAAAGGTGGATAC GGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAG CACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCT GAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATC CGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACC GCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCC CCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTG ATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCT TTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTG CCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTAC AATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATC CGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATAC CATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGT ATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTG AGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAG GCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGA CGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGC GCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTG AGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAG TCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTT CCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCAT CCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGT TTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC GTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCG CTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCA GATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACC TCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGA TAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACC TACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGG TATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTAT AGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAA AAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATC CCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGC AGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACA CCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTA CGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCC GGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAA CGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGT CCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCT GTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATG CTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGAT GCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGT AGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACG AAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGC TCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCA CGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGAC CAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCC AGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGA CAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGG GCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAG TCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGC CAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCA GCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGA GCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCG CATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATG GTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATAT TTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGA CCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTG GTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGC GGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGC TTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGA CGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCAC GCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCT GGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACA TTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTC CGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCAC CGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATAC CCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGG CGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATC CCGCGAAAT
 * Accepting Vector (pNIC28-Bsa4) with Insert (blaGES5)**


 * Figure 1.** Virtually cut pNIC28-Bsa4 (accepting vector for target gene kpblaGES5) with BsaI restriction enzyme. Displays the exact location where BsaI should cut pNIC28-Bsa4.

Dried gel and determined the purity and yield of YopH protein.
 * YopH Protein Characterization - Part 2 (6/30/2014) **


 * Figure 1.** SDS page gel of YopH samples from expression and purification. Lane 1 is the protein 1kb ladder. Lane 2 is the cell lysate before induction (sample 0). Lane 3 is the cell lysate after induction (sample 1). Lane 4 is the soluble fraction (sample 2). Lane 5 is the flow through (sample 3). Lane 6 is the wash (sample 4). Lane 7 is elution 1(sample 5). Lane 8 is elution 2 (sample 6).

Sample 0, 1, and 6 were all given DNA dye instead of protein dye. In addition, the protein dye was old and kind of chunky. When inserting the samples into the lanes, the samples would not sink to the bottom of their wells, some even traveling out and into other wells, explaining why the wells beside lane 1 and lane 8 appear to have samples when they shouldn't. Elution 1 and 2 (lanes 7 and 8 respectively) look very contaminated but elution 1 (lane 7) looks a lot like lanes 6 and 5 which is why it seems that the samples 3 and 4 migrated into wells 7 and 8. The gel is fairly unreliable and FPLC should just be used regardless to purify further.

__ WEEK 4 (6/23/2014 - 6/27/2014) __

Separated YopH protein samples 0 - 6 by gel electrophoresis and visualized with a dye.
 * YopH Protein Characterization - Part 1 (6/27/2014) **

Sample 0: Cell lysate before induction (500uL, from expression, 6/18/2014) Sample 1: Cell lysate after induction (500uL, from expression, 6/18/2014) Sample 2: Soluble fraction (50uL, from expression, 6/19/2014) Sample 3: Flow through (100uL, from purification, 6/25/2014) Sample 4: Wash (100uL, from purification, 6/25/2014) Sample 5: Elution 1 (100uL, from purification, 6/25/2014) Sample 6: Elution 2 (100uL, from purification, 6/25/2014)

Made an SDS-page gel for analysis and characterization of the purified YopH protein.
 * Made Homemade SDS-Page Gel Solutions for YopH Protein Characterization (6/26/2014)**

Purified the YopH protein using the His6 affinity tag and Ni-NTA resin.
 * YopH Protein Purification (6/25/2014)**

**Figure 1.** Amino acid 280nm nanodrop graph of elution 1 YopH trial 1, presenting 10mm absorbance vs wavelength nm. The absorbance value is 3.702.


 * Figure 2.** Amino acid 280nm nanodrop graph of elution 1 YopH trial 2, presenting 10mm absorbance vs wavelength nm. The absorbance value is 3.681.


 * Figure 3.** Amino acid 280nm nanodrop graph of elution 2 YopH trial 1, presenting 10mm absorbance vs wavelength nm. The absorbance value is 0.822.


 * Figure 4.** Amino acid 280nm nanodrop graph of elution 2 YopH trial 2, presenting 10mm absorbance vs wavelength nm. The absorbance value is 0.840.

Average absorbance values: Elution 1 - 3.6915 Elution 2 - 0.831

Molar Extinction Coefficient: 17670 Molecular Weight: 33381.6 g/mol

Beer's Law calculations: determined the concentration of YopH protein at a 280nm wavelength Elution 1 - 6.97mg/mL Elution 2 - 1.57mg/mL

Determined the YopH protein yield Elution 1 - 14.637mg (2.1mL) Elution 2 - 6.280mg (4.0mL)

Prepared an agarose gel to run and verify the following DNA pieces: amplified pGBR22 from PCR lab (6/20/2014; tubes A-D), stock pmCherry used in the submission of DNA sequencing (6/10/2014) and transformation of competent DH5alpha for midi prep (6/16/2014), and extracted pmCherry from midi prep (6/17/2014).
 * Agarose Gel Preparation (6/24/2014) **


 * Figure 1.** Gel lane 1: 1kb ladder 100bp, lane 2: PCR tube A with 0.3ng of pGBR22, lane 3: PCR tube B with 3ng of pGBR22, lane 4: PCR tube C with 30ng of pGBR22, lane 5: PCR tube D with no DNA (control), lane 7 with about 300ng of stock pmCherry, and lane 9 with about 300ng of extracted pmCherry from midi prep lab.

Ladder: ladder in lane 1 traveled too far down the gel compared to the other samples, indicating the wrong ladder may have been used.

PCR: Tube A (lane 2) showed no bands (may be because an insufficient amount of DNA was amplified). Tube B (lane 3) showed 1 band and tube C (lane 4) showed 3 bands. Theoretically, tube A-C should present the same number of bands in the same location in respect to their lanes. Since this is not the case, there may be contamination or not all of the PCR process worked (only parts of the DNA were replicated). Tube D (lane 5) showed no bands as expected because no DNA was present (control).

Stock pmCherry: With so many N's in the 1st DNA sequencing results, something was wrong with either the DNA or the primers. However, in the 2nd sequencing results, the UT' DNA sequencing facility's primers were used and there were still N's, meaning that the DNA had been degraded or contaminated. Based on the gel results (lane 7), a giant glob with streaks and 2 additional bands demonstrated that the stock pmCherry was indeed contaminated.

Midi prep pmCherry: The stock pmCherry used to transform the competent DH5alpha cells that produced the midi-prepped pmCherry plasmids had DNA sequencing results with a lot of N's like the midi-prepped pmCherry's DNA sequencing results. With that said, there may have been contamination in the stock pmCherry, hence contamination in the midi-prepped pmCherry. Based on the gel, the midi-prepped pmCherry (lane 9) had a giant glob with streaks and 1 additional band, similar to that of the stock pmCherry (lane 7). The giant globs of both lane 7 and lane 9 also were at the same location. This indicates that the midi-prepped pmCherry was contaminated, possibly due to the contamination of the stock pmCherry that transformed the competent DH5alpha cells.

** Analyzed DNA Sequence (6/23/2014) **
Determined where in the DNA sequence of the pGEMT plasmid that the purple protein sequence would be inserted to create pGBR22 (via computer lab).

WEEK 3 (6/16/2014 - 6/20/2014)
Amplified purple protein coding sequence in the pGBR22 plasmid using M13F forward primer and M13R reverse primer. Tube A had 0.3ng of pGBR22, tube B had 3.0ng of pGBR22, tube C had 30.0ng of pGBR22, and tube D had no DNA thus serving as a control. Replicated pGBR22 would be verified by an agarose gel.
 * PCR for pGBR22 (6/20/2014) **

Produced YopH protein from transformed BL21(DE3) bacterial cells. Obtained a pellet mass of 2.86g. Would be later purified and characterized for enzyme assays.
 * YopH Protein Expression (6/17/2014 - 6/19/2014)**

Extracted pmCherry from transformed DH5alpha bacterial cells.
 * Midi Prep for pmCherry in DH5alpha (6/17/2014)**


 * Figure 1. ** Trial 1 nanodrop graph of extracted pmCherry, displaying 10mm absorbance vs wavelength mm. The concentration registers as 167.3ng/uL.


 * Figure 2.** Trial 2 nanodrop graph of extracted pmCherry, displaying 10mm absorbance vs wavelength mm. The concentration registers as 173.6ng/uL.


 * Figure 3.** Trial 3 nanodrop graph of extracted pmCherry, displaying 10mm absorbance vs wavelength mm. The concentration registers as 173.2ng/uL.


 * Figure 4.** Trial 4 nanodrop graph of extracted pmCherry, displaying 10mm absorbance vs wavelength mm. The concentration registers as 173.9ng/uL.

Average concentration of the extracted pmCherry was 172.0ng/uL.

>xbgMPsp6-Sp6 838 838 0 0.05 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNNNNNNCCCTNNNAANGNNNNNNTNNNNNNNANNNNNNNNNNNNNNNTNTGGNANNNNN NNNNNNNNNGNNNNNNNTNTNNNNNGCNNNGNNNNNCTGCNCNGNNNNNNNGNNNNNNTNNNNNNNCNNNNNNNCCNNTANNNANNGNNCGCNNTNNTTNNNCNTNNNNNNNNN NTNNNTCNCNCCCNANNNGGNGCCNGNNNTNGAGNACANCCNNNNNNCGGNNGCCTNCCNGCCCANNGNCTTNTTCNNCNTTCNNGGGGCGNCNGAGGGGAAGATNGTGCCNC NCNNNTTNNNNNNNNNNNNANCTNNNNNNNNTNNNNNNNNNANNNNNNNGGGNGNNNNNNNNNNNNNNNNNNNNNNAANNCNTCNCNNNNNNNCNCNTGNNGNNNTNNNNNANN NNNNNNNNNAANTNNNCNGNNATNTCNNCNNNNAGCNNCGCGNANNNCTTNNNNTCGTNCTNNNNNTGAGGNGANNNGNTNNNNCNNNCNANGNNNNNCNNNTANACTTTGGGAT NNNNNNNNNNTCNNCTNNTTCTNNNNNNTTGCGNCGNNNNTTNACNNNCNNCNCNNNCNCNNANNNNNGNNAGGNTNNNNNNNNNNNCCNNGNNNNCNNNNNNNNNTNNNANNNN NNNNNNNNNGNTCCNNNNNNNNNTNNNNNNNNGNNGNNCCNNNNNCTGCNNNNNGTNNNNNTCNTNNANNANTGAAANNNNNNNGANGNNNNCCTNNAANAACAANNNNNNNNN NGNNNTNNNTNNNNTNNNNNANTACCTACNNNNNCCNACN
 * Figure 5.** DNA sequencing results of pmCherry, using sp6 forward primer (requested in-house primers), from the UT Core ICMB (DNA sequencing facilities). Too many N's indicated uncertainty.

>xbgMPt7-T7 847 532 1 0.05 NNNNNNNNNNNNNNNNNNNNNNCNNNNNNNNTTCNANAGGNNCNNGAGGNTCNNNNNNTNTNNNNNNAANNNNNNNNNNNNNNNNCNNNNNTTCTANNNAANATNNNGNNNNNTT NNNCNANNGTGGTTGGTCTTCNTTTNAGCNNGNGNCTGNTNNANTTNCTGANTGNNNNGNANCNNNNNNNNNGGNNCNNNCNNNNNNNNNTGANTGNNCNNNTCCTGGTTTTGCTTT ANGTNCNNNNNCNNGNNGNNNCNGGNCTTGATCTNATGTNNCANACNTCTGNNTGCNAGCTNAANGTCCNNNCNCCNTTNCNNNNGGNGNNNNNNNNNNANATNNNGNNCCNNNN NNNTCNNANTNTGGNNNNNCTCTTTCCTGTTGNCCAAANNCNACNNNGGGANAANTNNNTTGNTNNNNTCNGGNAAACCCNNNNCNNGGACNNANTGCTGGAANTCNACCTCTGAN ANNNNANNCTTNNAGCTCTCNNNNANCNNANAGGTGAGCCCCTCNACAGCTTGNNNAGTTGNNGTGCNGCTAACCTCTNNCTNCCTTGCNNCTCCNTCANANACTTTGGGATTGGA ACCNNNNNNNNNNNATTCTNNNNNNNTGCGNCCANATTCACCAACNNCNNNGCCGCCCNCNNNAGTNAGGNTCTGCNNANGTTCCCTGNGCNNNNNNCTNANNNNNAGNANNNN GNNGAANGNCTCCNTCTTNAAGTTAANCNNNGNNGCCCCCNAGCCTGCTNNNNTGTNNNACNTGTTGATNNNTNNNNTNNNNTNGANGGNNTNCTGNNNAANAACANNTATCNNGT ANNNNATNNTGNNNNNNANNNNNNGCTNGNNCCNACNN
 * Figure 6.** DNA sequencing results of pmCherry, using t7 reverse primer (requested in-house primers), from the UT Core ICMB (DNA sequencing facilities). Too many N's indicated uncertainty.

Transformation of DH5alpha bacteria with pmCherry plasmid (with ampicillin resistance; from transformation efficiency lab on 6/12/2014) to make more pmCherry.
 * Transformation of Competent DH5alpha Cells for Plasmid Prep (6/16/2014)**

Prepared 20 plates for culturing bacteria with resistance to kanamycin. Would be later used as the pre-step to protein expression.
 * Prepared LB Agar + Kanamycin + Sucrose Plates (6/16/2014)**

WEEK 1 & 2 (6/5/2014 - 6/13/2014)

 * Transformation Efficiency Results of DH5alpha with pmCherry and Ampicillin (6/12/2014)**


 * Figure 1.** DH5alpha transformed bacteria with 1ng of pmCherry plasmid (from tube A). Transformed bacteria grew on LB agar plates with ampicilllin (AMP) for an approximately 24-hour period in 37C incubator. Transformation efficiency about 3000ng/uL.


 * Figure 2.** DH5alpha transformed bacteria with 5ng of pmCherry plasmid (from tube B). Transformed bacteria grew on LB agar plates with ampicilllin (AMP) for an approximately 24-hour period in 37C incubator. Transformation efficiency about 81ng/uL.


 * Figure 3.** DH5alpha transformed bacteria with 25ng of pmCherry plasmid (from tube C). Transformed bacteria grew on LB agar plates with ampicilllin (AMP) for an approximately 24-hour period in 37C incubator. Transformation efficiency about 52ng/uL.

**Figure 4.** DH5alpha bacteria (not transformed; no plasmid; control) from tube D. Bacteria was placed on LB agar plates with ampicillin (AMP) for an approximately 24-hour period in 37C incubator, but did not grow/ form any colonies. Transformation efficiency was 0ng/uL.

Prepared 1.7mL centrifuge tube of pmCherry (plasmid) with primers sp6 & t7. A coreweb.icmb username and password was created. Tube was submitted to UT DNA sequencing.
 * Submitted pmCherry for DNA Sequencing Facility (6/10/2014) **

>XBG-Sp6 676 676 0 0.05 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCTNNNGACNNNNNAANANANNNNNNNNNGATNTNNANAANATTNNNNNNANANNNNNANNNNNANNAAANNNNNTNGGNNNN NNNNGNTTCNNNNNGNNGCTCNNNNNNNNNGTGNNNNNNNNNNANNGGNGGNNAAAACNTCNCNCTNCCCCCNGNTAAATNANNNGCNNNNNNNNGNNNGCAAGGNNNGATCC CCCNACNNCNACNANGGNNNNATCCCNNNNNNNNANNANTGANNNNNNNNNNNNNNNNNNNNNNNTNTNNNNNNTTNNNNNNNNNNNNNNNNNNNNNGNNNTATAAANNNNNTA GNTCCTTNTNNNCTNNTNNNANNCNCNNNTNNNTTGCNTTNNNNNNNGNNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNTNNNNNNNNNNNTTNCTGTNNNNNNANNNN NNANNTNNNNNNNTTTNNCANNCNNNNNTGTGNNNTGNNNANNNTGNNNGNNGNNNTCCCNNNNGGGNNNNGNNNNNNNNCNTCNACNTNNNNNNNNNNNNTNGNATTNTNNNNN NCCTNCTNNNGNATGANNTNNNNNANNTCNNCNNNGCCTGTCNN GNNNNNNNNNNNANNNNNNNNNNNNTGNNNNNNNNNNGANNNTNNCNNGNTGGCNNGNNN
 * Figure 1.** DNA sequencing results of pmCherry, using sp6 forward primer, from the UT Core ICMB (DNA sequencing facilities) - run 1. Too many N's indicated uncertainty. DNA sent again without primers (requested in-house primers) for another sequencing run.

>xbgVsp6-Sp6 1023 1023 0 0.05 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNNNNGNCNNNNNANNNNNNNNNNNNNNNNNNNNNCNNNNGGACCNTNNGNNNNNNNNTNNN NNNNGGGNCNNTNNTNNNGGNGNANGNGNGNNNNNNNNNGNNNNNNGGGNNNNANNNNNGGNCNNNCNCCNNTNNNNNNGNNCNGCTGGNNNTGNNCNGNNANCTGANCNNN GNCNCCCNNNNNNNGGNGNCNNATNTNGANNNNAACCNNNNNNCCGCNNTTTTNNTNNTCNGNNNNNNNATCNACTTTNNGGNGGNNNNNGNTGGANNNNNNNTGGNGCNCNNN NATGTNNCCGCCCCCNNNNNNNTCNNNNCCCTNNNNNNNNNNNCGGNNNNNAGGGNGNCNNANNNNANNTNAANCCNNNNNCNNNNNNNNNNNNNNCCNNTNNCCTCNCNNANN ANGGTGNNCCCNNGGANNNNNCCNCNNNNCNNGNNGNANTNNNNNANNTCNTGNTGNTNNNNNNGNNNNNNNATGTNNNNNNNNNACTGNNNNNNNNANNNNNNTGNNNNNAGA AANNTNGNGNNCGGGNTANNGNNANNTTGCNGGGGNGANNNANNNNNNCNNNNGGNNNNNNTNNNNNNNNGGGNNNGNNNNNNNNCNNNNNNNNACNNNTNNANTTNGNNNCN NNNNNNNNGCNNNNNNNNNNNNTNANNNGNNANNNANNCNNNNNNNNNNNNNGTNNNTNNTNCNCCGNGANTGAAACNNNTTTNNNNNNANANTGNNGNNCNNNNTNNNNCNNN NTNCNNNNNNNNNNNNNTNCAGCNNNNNNTNCTNNNANTTNCANCNNCNNNNNGCNANNCGNGNNNNNNNNNNNNNNNNNNNNNNNANNNACNNGNANNNNNNANNNNNNNNNN TNNNNNNCNNNNNNCNNGGNNNNNNNNNNNNNCTNNCNTNGNNNGNANNNNNNGNNNTNNNNTCANNNNNNNNNNNNANNGANNNNNNGNNNNNNNNNCAGNNNNCCNNNNNN NN
 * Figure 2.** DNA sequencing results of pmCherry, using sp6 forward primer, from the UT Core ICMB (DNA sequencing facilities) - run 2. Still too many N's (indication of uncertainty). However, there appears to be more nucleotides detected and recognized (6/19/2014).

Searching for information regarding potential targets for VDS.
 * Target Discovery (6/6/2014 & 6/9/2014) **

diluted primer pLIC-rev for DNA cloning.
 * Primer Dilution of pLIC-rev (6/5/2014) **