Renee+F.

=FALL 2013=


 * WEEK ELEVEN & TWELVE**
 * (11/13/13)** Cells were lysed by sonication, purified, and characterized.
 * (11/8/13)** Large culture was prepared, induced, re-suspended, and stored in Lysis Buffer.
 * (11/7/13)** Transformation results checked and a small culture started
 * (11/6/13)** Transforming new surrogate enzyme into BL21(DE3) cells


 * New Surrogate Clone Nanodrop**


 * Results of Transformation From 11/7/13**


 * Characterization Results Before Drying**


 * WEEK NINE & TEN**
 * (10/23/13)** Virtual screening- NADPH control dock and control library dock
 * (10/23/13)** Master plate and mini-prep
 * (10/24/13)** DNA sequencing
 * (10/25/13)** Virtual screening- NIH dock
 * (10/26/13)** Primary and secondary PCR
 * (10/30/13)** Secondary gel extraction
 * (10/31/13)** PCR squared off of secondary gel extract
 * (11/1/13)** pNIC-Bsa4 grown up


 * PCR squared off of secondary extract**



A gel extraction was preformed on secondary PCR to then continue onto PCR squared. This was done in hopes of retaining high concentration values and purity.
 * Secondary gel extraction**

Primary and secondary PCR was preformed again since the DNA sequencing results came back negative. This time, the protocol was altered in order to optimize the results.
 * Primary and Secondary PCR**

Since the results from the NADPH control dock were good, I decided to run a small library of ligands (~550).
 * Virtual screening-NIH dock**

The results from DNA sequencing (sent 10/24/13) were not indicative of a positive clone. Again, only the beginning ~200 nt and the ending ~200 nt were cloned into pNIC-Bsa4. However, this phenomenon is random (sometimes only beginning clones in for example). We have not been able to conclude a reasonable explanation, but cloning will be attempted again.
 * DNA sequencing results**

A master plate was made off of the transformed cloning from 10/18/13 and mini-prepped to be sent to DNA sequencing.
 * Master Plate**

Tony was able to obtain a crystallographic structure of L. major DHFR-TS from Dr. David Matthews (we came across references to this structure in literature about our target). A control dock on this new structure was performed and the results were promising (higher than our homology model).
 * Virtual Screening- NADPH and control library dock**



Nice work in the wet lab Renee. Good captions and analysis. Do you have any virtual data? Thank you. -Max 10/21/13 Overview
 * WEEK SEVEN & EIGHT**
 * (10/7/13)** Transformation results from 10/5/13 were checked. Secondary PCR was made and ran on a gel.
 * (10/8/13)** Virtual Screening- control dock of NADPH.
 * (10/9/13)** Virtual Sreening- positive and negative control library dock.
 * (10/8/13-10/9/13)** Sending cloning results to DNA sequencing.
 * (10/14/13)** Two rounds of PCR squared and cleanup.
 * (10/17/13)** Gel Run of Clean PCR. Cut pNIC-Bsa4.
 * (10/18/13)** Cohesive End Generation and Transformation.
 * (10/19/13)** Transformation Results were checked.


 * Transformation Results from 10/18/13**

Protocol was carried out almost verbatim from the protocol except three transformation tubes were plated instead of two and the E. coli was heat-shocked for 45 seconds instead of 30 seconds. The three transformation tube concentrations were as follows: Tube A: 2ul of vector and 4ul of insert Tube B: 5ul of vector and 10 ul of insert Tube C: 20 ul of vector and 17ul of insert
 * Cohesive End Generation and Transformation**


 * Gel Run of Clean PCR and PCR squared from 9/6/13**

pNIC-Bsa4 from the previous two weeks was midiprepped, cut, and then cleaned up. The following are Nanodrop concentration from before and after cleanup. I modified the preparation protocol because 2.25 ng of my plasmid was about 40ul, which was over the final volume. I double the final concentration, and subsequently doubled the amount of reagents. Also, I let it cut for 3 hours.
 * Cutting the pNIC-Bsa4**

Two rounds of PCR squared were preformed off of secondary PCR from 10/7/13. One sample was cleaned and the other was run on a gel. However there were bands of contamination.
 * Two Rounds of PCR squared and Cleanup**

The results indicated that only parts of our gene cloned into the pNIC-Bsa4, the beginning 100-300 nt and the final 100-300 nt were clonning into our gene.
 * DNA Sequencing Results**

For both control docks I used the 3inv homology model and superimposed 3inv to get the conformation of NADPH/the ligand and define the binding site. For the NADPH control dock this created a clash between NADPH and the homology model. However, this did not happen when I defined the binding site using NADPH and C50 (the ligand complexed into 3inv). Results of NADPH dock: Results of Control dock:
 * Virtual Screening-NADPH and Control Library Control Dock**
 * Secondary PCR**

There were three very large colonies. Each colony was mini-prepped, nano-dropped, and sent to sequencing.
 * Transformation Results from 10/7/13**

Overview
 * WEEK FIVE & SIX**
 * Good data but try to add captions and analysis. Thank you. -Max 10/07/2013
 * (9/25/13) Two round of secondary PCR were run.**
 * (9/27/13) Positive and negative control ligands were found and 3D SDF files were concatenated.**
 * (10/2/13-10/4/13) New pNIC-Bsa4 was grown up to be used for cloning.**
 * (10/4/13) Four rounds of PCR squared were run and then extracted to use for cloning. pNIC-Bsa4 was also midi-prepped.**
 * (10/5/13) Cohesive End Generation and Transformation**

Secondary PCR

Positive and Negative Control Ligands
 * Positive control ligands found via PDB structure similar to our target protein (ALL above 50% identity) and seeing what the protein was complexed with. The following compound identifiers were obtained as 3DSDF files from PubChem and then concatenated on the DDFE. **
 * 1) Natural Substrate= DHF **
 * 2) 1CX **
 * 3)UMP **
 * 4)C50 **
 * 5)MTX **
 * 6)TMQ **
 * 7)2CY **
 * 8)DQ1 **
 * 9)WRA **
 * 10)P128 **
 * 11)P65 **
 * Negative control ligands (except for aspirin) were obtained by finding structures with similar physio chemical properties as the positive controls. All the compounds were obtained as 3DSDF files from PubChem and then concatenated on the DDFE. **

Growing up more pNIC-Bsa4

Four Rounds of PCR squared






 * Concentration of pNIC-Bsa4 after midi-prep**

WEEK THREE & FOUR great work Renee - keep driving fowrard with the cloning - we need to get your clone soon so that you guys can move forward - Dr. B 100113 Overview
 * (9/11/13) Research was resented in class.**
 * (9/13/13-9/14/13) New pNIC-Bsa4 was attempted to be grown up in a large-scale expression to be used for cloning. The attempt was unsuccessful probably due to the use of old colonies.**
 * (9/16/13-9/19/13) Homology models were made.**
 * (9/17/13) pNIC-Bsa4 grown during the summer was prepared for cloning.**
 * (9/18/13) A gel was run to ensure that the pNIC was successfully cut the previous day.**
 * (9/19/13) Cohesive end generation and transformation was done. Four rounds of PCR squared were preformed. **
 * (9/20/13) Transformation was unsuccessful. The four rounds of PCR squared were check on the gel, extracted, and then cleaned up due to low purity values. The resulting concentration was not very promising.**

Homology Model

pNIC-Bsa4 Used for Cloning

Virtual Gel

Cut pNIC-Bsa4

PCR Squared

PCR Squared Gel Extraction Nanodrop Results (Before Cleanup)

PCR Squared Clean Gel Extraction Nanodrop Results.

WEEK ONE & TWO Overview
 * Renee - good work. Dr. B 090913**
 * (9/2/13) New tail primers were ordered, a new oglio-mix was prepared. Primary and secondary PCR was preformed and tested on the gel. The results were a smear on primary PCR and a semi-bright band on the correct nuceotide position (1500nt).**
 * (9/6/13) PCR squared was preformed and tested on gel.Then gel extraction was performed on the PCR squared product since the band was at the correct position. The concentration after gel extraction was around 50ng/microliter. The 230nm. peak was high while the 260 nm. peak was low, which could account for poor cloning, but I have decided to continue to cloning next week.**

Nanodrop Concentration after Gel Extraction

Gel Extraction

PCR squared

Primary and Secondary PCR

NEB 1 KB Ladder

WEEK FIVE

Primer Design (Putative acid phosphatase of H. pylori in pNIC-Bsa4)
 * __Forward Primer:__**

GC Content 41.2%
 * 5’ TACTTCCAATCATGGTAAAAAAGACGCTGGCATC 3’ 34 bp**
 * 0 mM Mg2+ Tm 62.1 oC 1.5 mM Mg2+ Tm 69.6 oC 2 mM Mg2+ Tm 70.1 oC**
 * 4 mM Mg2+ Tm 71.1oC 6 mM Mg2+ Tm 71.6 oC**


 * __Reverse Primer__:**


 * 5’ AAAGCGTGGCAGAACAAAAAGTAACAGTAAAGGTGGATA 3’**


 * __Reverse complement it:__**

GC Content 39.5%
 * 5’ TATCCACCTTTACTGTTACTTTTTGTTCTGCCACGCTT 3’ 38 bp**
 * 0 mM Mg2+ Tm 63.7 oC 1.5 mM Mg2+ Tm 71.5 oC 2 mM Mg2+ Tm 71.9 oC**
 * 4 mM Mg2+ Tm 72.9 oC 6 mM Mg2+ Tm 73.3 oC**

WEEK FOUR

Protein Purification: (PfDXR in pNic-BSSA4)

SDS-PAGE Gel Results:

WEEK THREE

Third PCR: (pmCHERRY)

Large-Scale Protein Expression: ( PfDXR in pNic-BSSA4) pellet weight=2.8 g.

Second PCR: (pgbr22)

_ WEEK TWO

First PCR Results:

Midi-Prep DNA Sequence: (92015)
 * NNNNNNNNNNNNNNNTNNACTTTAGANGAGATNTACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATNTGGGT**


 * ACCGAGAACCTGTACTTCCAATCCATGGAGACCGACGTCCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGAT**


 * ATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGA**


 * AAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGNAGTCTGCAAATCCTTTTATGATTTTCTATCAAACANAAGAGGA**


 * AAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAATAGTAANTCNNGCGGGTTTGTTNNNGANNAA**


 * GNNNGCAANANNAAANANGTGTNNNTNNAAAAGCGNNTTNTTTTNNGTNNNNACNNNNNCATCNCNNNNNTNCTTTNNNN**


 * NTTGCNCCANTGNNNNNTTNNNNNNNNNANNANAAGNNNNNNNNNNNNNNNNNNNN**

Midi-Prep Nanodrop:

___ WEEK ONE

DNA sequencing: ( 91623 )


 * NNNNNNNNNNGNNNNNATAGAATACTCAAGCTATGCATCCNACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCG**


 * GCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATAT**


 * GTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAA**


 * AGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCA**


 * TTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAA**


 * CTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCT**


 * CTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTT**


 * GCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAA**


 * ATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACA**


 * ACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCAC**


 * TAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCAC**


 * TGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTC**


 * GCCAGCTGGCGTAATAGCGAANANGCCCGCACCGATCGCCCNTNCCCAACAGTTGCGCANCCTGAATNGNNAATGGACGC**


 * NNCCNNNANNNNNNCATTNAGNNNGNNNNNNNNNNNNNCNCNCAGCNNGACGCNNNNNNTTNCNNCNNNNNNNNCCNNNT**


 * NNTNNNNNTTCNNNCNNNNTNCNNNNCNCNNNNNNNNNNTNNCCNNNANNNNNNNNNGGNNNNNNNTNNGNNNCNNNNNN**


 * NNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN**

Plasmid ** : Plate A: 1080 colonies / 2ng plasmid = 540 colonies per ng plasmid (pNIC BSA4) Plate B: 456 colonies / 10ng plasmid = 45.6 colonies per ng plasmid (pNIC BSA4) Plate C: 68 colonies / 50ng plasmid = 1.36 colonies per ng plasmid (pNIC BSA4)
 * pNIC-BSA4 || Renee F. & Young L. || TBD || E.coli DH5alpha || 160mL ||
 * Transformation Efficiency calculation: **




 * Primer Dilutions:**