Jensen+G.

Week 15&16 __**Enzyme** **Assay**__ Enzyme assay with stock YopH enzyme was performed.

Figure 1: Results of enzyme assay from 234uM sample of YopH enzyme.

Sample Absorbance at 410nm A (no enzyme) 0 B (10ng enzyme) 0.054 C (20ng enzyme) 0.422 D (40ng enzyme) 0.771 E (60ng enzyme) 1.020 F (80ng enzyme) 0.817 G (100ng enzyme) 0.853 H (120ng enzyme) 0.827

__**Protein Expression**__ Tubes in protein expression were accidentally left in the shaker and were too saturated to proceed. Step one (day one option A) was repeated. Large culture step: The OD600 number was monitered until it reached .5 (which sadly never happened)

Measuring OD values - 23uL of total culture

Time: Absorbance at 600nm

10:53 .052

11:32 .064

11:56 .072

1:02 .108 <-- Culture was added until here

1:46 .207

1:55 .350

2:05 .361

2:13 .395

The OD600 value never reached .5 (within 1-2 hours) because it was probably out of the log phase.

Week 13&14 __**Results and Resubmit to DNA Sequencing**__ I sent to DNA sequencing twice, first with forward primers: NNNNN
 * 1:**

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
 * 2:**


 * 3:**
 * NNNNN**


 * 4:**
 * NNNNN**

The results were not a clone (not anything really), but because my master plate did grow and it was evident something was there, so I resubmitted to DNA sequencing, this time with reverse pLIC primers.

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNNNNNNNANTNNNNNNNTNNNNNNNGANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANTNNNNNNGNNGANANNNNNNCCGCNNNNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNCNNCNCCCNNCCNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNN
 * 1:**

NNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNTNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNNNNNNNNNNNNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
 * 2:**


 * 3:**
 * NNNNN**


 * 4:**
 * NNNNN**

The results were not much better, so the move to a surrogate (YopH) was made.

__**Protein Expression (YopH)**__ Because Both attempts to get a positive clone from sequencing with PP2C were inconclusive, protein purification was continued with YopH.

The YopH enzyme was transformed into BL21(DE3) cell.

The Small culture+overnight induction (option A) was started: 4 ml LB + 1 colony+ 50 ul Kan were innoculated in a tube, this was repeated for 4 different colonies.

Week 11&12 __**Cloning**__ Figure 1: Sample 1 Figure 2: Sample 2 Figure 3: Sample 3 Figure 4: Sample 4
 * Miniprep:**

__**Submitting to DNA Sequencing**__

Week 9&10

__**Cloning**__ Pnic-Bsa4 was successfully cut and the PP2C gene was inserted. Miniprep has not yet been performed and nothing has been sent to sequencing.

Figure 1: Master plate of PP2C in Pnic BSA4 Figure 2: Miniprep of cut pNICBSA4.


 * I grew more pNIC-BSA4 and made stock kan this week also*

Week 7&8


 * __MidiPrep__**

Nanodrop results of the midiprep from our culture of pNICBsa4. The concentration is not so great, and it has some contamination. Cloning will be continued with this, but more pNIC will be grown up in the meantime.


 * __PCR Cleanup__**

Figure 1: Nanodrop of the PCR2 of our gene PP2C. This has a good concentration with very little contamination. Yay!

Nice images and captions. Try to include a bit of analysis on what you're doing though. Keep it up! - Michael T. Week 5 & 6

__**Transformation**__

Figure 4: Overnight growth of pNIC-BSA4 in LB+Kanamycin media. Figure 3: pNIC-BSA4 pellets from centrifuging overnight growth.

__**PCR Squared**__

Figure 2: Agarose gel run in 1x TAE for T. Gondii PP2C, Lane one was skipped, lane two was a 100bp ladder (shown right of image), lane three was sample 1 of PCR squared, lane four was sample 2 of PCR squared, lane five was sample 3 of PCR squared, and lane six was sample 4 of PCR squared. Because this PCR was just amplifying secondary PCR, a bright band would be expected at the gene length (1200bp). All samples showed a band at this length except sample 1.

__**Secondary PCR**__

Figure 1: Agarose gel run in 1x TAE for T. Gondii PP2C, Lane one is represented with a 100 bp ladder (image on right). Lane 2 is the secondary PCR mixture. A band appeared at the gene length (~1200 bp) so we went on to PCR squared.

Week 3 & 4 Jensen - need your tail primer design info, also use more formalized captions. Include ladder image -- 092713 - Dr. B __**Primary PCR**__



Gel produced from run one of primary PCR on a 1x TAE gel (T. Gondii PP2C). Lane 1: skip, lane 2: 100 bp ladder, lane 3: Sample including Primer Mix. A smear appeared as expected in lane 3.


 * __Primer Overlap (Primer Mix)__**

1 ul of each one of our oligos (30 total) were added to a 1.7 ml tube. 70 ul of autoclaved water was added to this (Total volume=100ul). Primer mix was properly labeled and stored in -20 C feezer.

__**RE Digest**__

Gel produced from RE Digest (pGBR22) on 1x TAE Gel. Lane 1: Skip, Lane 2: 1 kb ladder, Lane 3: No RE, Lane 4: No RE, Lane 5: Eco RI, Lane 6: Pvu II, Lane 7:Eco RI and Pvu II. Only lane 6 showed plasmid fragments, meaning samples in lanes 5 and 7 were not cut by the restriction enzyme as they should have been.

Week 1 & 2

__**PCR with pGBR plasmid**__

Here you can see the gel result from my PCR run. Sample B appeared nicely at about 900-100bp, sample A had no bands, and sample C had two bands, one at the correct length and another larger one, suggesting too much plasmid was used.

__**Primer Design**__


 * The amino acid sequence of T. Gondii Protein Phosphatase 2C was input into DNAWorks:**

 1 MADAKTLLGKVKRATGMGVGEGPSVAKKPKYTATVPGFTPPSGDQRMNEFMAVDTSGEFM  61 RHLYIEEGRTVCASATSRNRRPTSESPHSDDVVVVEGMLRGRPETRVHAMFDGFQGRHSA  121 MWLAQNVMNYLNDLRDVNEEEITRQFERMDGDLRAANLPGGSSALIIFVRYEKKPTEARV  181 VGRQIVPEGAKEFTSVAEALGGPLMPVVAMNFRRDPRAAKGIYTIHVASLGNSRCVLKSG  241 RTAIHLSTPHTASSHKERHRVQAAGGVFTTVNGELLLGGVVPMTRAFGSFDFKKGGQGKL  301 QQDLVSAVPDVTTFFAYPGDDIVAGTAGAFAHFRSHAAIAAAIALYPVSPETVLDAAKAM  361 VVNAKRRKVTKNISTFVRHLPESRTRSQKMLEGTSGENGEX


 * The following DNA sequence was ouput based on the amino acid sequence: **

 1 ATGGCGGATGCTAAAACCCTGCTCGGTAAAGTTAAACGTGCGACCGGTATGGGTGTTGGT

 61 GAAGGTCCGTCTGTTGCGAAAAAACCGAAATACACCGCGACCGTTCCTGGCTTTACTCCG

 121 CCGTCTGGTGACCAGCGTATGAACGAGTTCATGGCAGTTGACACCTCTGGCGAATTCATG

 181 CGCCACCTGTACATCGAGGAAGGTCGTACCGTTTGCGCGTCTGCGACTTCTCGTAATCGT

 241 CGCCCGACCTCTGAATCTCCGCACTCTGACGATGTTGTTGTGGTTGAGGGTATGCTGCGT

 301 GGTCGTCCGGAAACCCGTGTTCACGCGATGTTCGACGGTTTCCAGGGTCGTCACTCTGCG

 361 ATGTGGCTGGCGCAGAACGTTATGAACTACCTGAACGACCTGCGCGACGTCAACGAGGAG

 421 GAAATTACCCGTCAGTTTGAACGCATGGACGGTGATCTCCGTGCAGCGAACCTGCCGGGT

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 481 GGCTCTAGCGCCCTGATCATCTTTGTTCGCTACGAAAAGAAACCAACCGAAGCGCGTGTA

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 541 GTTGGCCGTCAGATCGTCCCGGAGGGTGCGAAGGAATTCACTTCTGTGGCGGAAGCGCTG

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 601 GGCGGTCCACTGATGCCTGTCGTTGCGATGAACTTCCGTCGTGACCCGCGTGCTGCGAAA

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 661 GGTATCTACACCATCCACGTAGCGTCTCTGGGTAACTCTCGTTGTGTACTGAAATCTGGC

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 721 CGTACCGCGATCCACCTCTCTACCCCGCACACTGCCTCTTCTCACAAAGAACGCCACCGC

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 781 GTACAAGCCGCTGGCGGTGTCTTTACGACGGTAAATGGTGAACTGCTGCTGGGTGGTGTT

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 841 GTCCCGATGACCCGTGCGTTCGGTTCTTTCGACTTCAAAAAAGGCGGTCAGGGTAAACTG

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 901 CAGCAGGACCTCGTTTCTGCGGTTCCTGATGTTACCACCTTCTTCGCGTACCCAGGCGAT

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 961 GACATCGTTGCAGGTACTGCGGGTGCTTTCGCGCACTTTCGTTCTCATGCCGCAATCGCT

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">1021 GCGGCTATTGCCCTGTATCCGGTTTCTCCTGAGACTGTTCTGGACGCCGCAAAGGCTATG

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">1081 GTTGTTAACGCGAAACGTCGCAAAGTTACCAAAAACATCTCTACGTTCGTTCGTCACCTC

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">1141 CCGGAATCTCGTACCCGTTCCCAGAAAATGCTGGAAGGTACTTCCGGTGAAAACGGCGAA

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">1201 TAA


 * This was found under the following parameters:**

Total Size Of Gene ......... 1203 nt Protein Residues ........... 401 Mutatable Residues ......... 385 Fixed Nucleotides .......... 48 nt Degenerate Nucleotides ..... 0 nt Oligo Size ................. 60 nt Annealing Temp ............. 62 +/- 1*C Oligo Concentration ........ 1.00E-7 M Sodium Concentration ....... 5.00E-2 M Mg2+ Concentration ......... 2.00E-3 M Codon Frequency Threshold .. 10% Repeat Threshold ........... 8 nt Mispriming Threshold ....... 8/18 (6 exact) nt


 * <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">From all of this information the following 30 oligonucleotides were formed: **

<span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 1 ATGGCGGATGCTAAAACCCTGCTCGGTAAAGTTAAACGTGCG 42 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 2 TTTCGCAACAGACGGACCTTCACCAACACCCATACCGGTCGCACGTTTAACTTTACCGAG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 3 GGTCCGTCTGTTGCGAAAAAACCGAAATACACCGCGACCGTTCCTGGCTTTACTCCGCCG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 4 AGGTGTCAACTGCCATGAACTCGTTCATACGCTGGTCACCAGACGGCGGAGTAAAGCCAG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 5 GTTCATGGCAGTTGACACCTCTGGCGAATTCATGCGCCACCTGTACATCGAGGAAGGTCG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 6 CGGGCGACGATTACGAGAAGTCGCAGACGCGCAAACGGTACGACCTTCCTCGATGTACAG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 7 TCTCGTAATCGTCGCCCGACCTCTGAATCTCCGCACTCTGACGATGTTGTTGTGGTTGAG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 8 CGCGTGAACACGGGTTTCCGGACGACCACGCAGCATACCCTCAACCACAACAACATCGTC 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 9 AAACCCGTGTTCACGCGATGTTCGACGGTTTCCAGGGTCGTCACTCTGCGATGTGGCTGG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 10 TTGACGTCGCGCAGGTCGTTCAGGTAGTTCATAACGTTCTGCGCCAGCCACATCGCAGAG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 11 ACCTGCGCGACGTCAACGAGGAGGAAATTACCCGTCAGTTTGAACGCATGGACGGTGATC 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 12 TGATCAGGGCGCTAGAGCCACCCGGCAGGTTCGCTGCACGGAGATCACCGTCCATGCGTT 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 13 GCTCTAGCGCCCTGATCATCTTTGTTCGCTACGAAAAGAAACCAACCGAAGCGCGTGTAG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 14 AAGTGAATTCCTTCGCACCCTCCGGGACGATCTGACGGCCAACTACACGCGCTTCGGTTG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 15 GGGTGCGAAGGAATTCACTTCTGTGGCGGAAGCGCTGGGCGGTCCACTGATGCCTGTCGT 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 16 ATACCTTTCGCAGCACGCGGGTCACGACGGAAGTTCATCGCAACGACAGGCATCAGTGGA 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 17 GCGTGCTGCGAAAGGTATCTACACCATCCACGTAGCGTCTCTGGGTAACTCTCGTTGTGT 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 18 CGGGGTAGAGAGGTGGATCGCGGTACGGCCAGATTTCAGTACACAACGAGAGTTACCCAG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 19 GATCCACCTCTCTACCCCGCACACTGCCTCTTCTCACAAAGAACGCCACCGCGTACAAGC 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 20 CCAGCAGCAGTTCACCATTTACCGTCGTAAAGACACCGCCAGCGGCTTGTACGCGGTGGC 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 21 AATGGTGAACTGCTGCTGGGTGGTGTTGTCCCGATGACCCGTGCGTTCGGTTCTTTCGAC 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 22 ACGAGGTCCTGCTGCAGTTTACCCTGACCGCCTTTTTTGAAGTCGAAAGAACCGAACGCA 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 23 CTGCAGCAGGACCTCGTTTCTGCGGTTCCTGATGTTACCACCTTCTTCGCGTACCCAGGC 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 24 GAAAGTGCGCGAAAGCACCCGCAGTACCTGCAACGATGTCATCGCCTGGGTACGCGAAGA 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 25 GTGCTTTCGCGCACTTTCGTTCTCATGCCGCAATCGCTGCGGCTATTGCCCTGTATCCGG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 26 AACCATAGCCTTTGCGGCGTCCAGAACAGTCTCAGGAGAAACCGGATACAGGGCAATAGC 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 27 GCCGCAAAGGCTATGGTTGTTAACGCGAAACGTCGCAAAGTTACCAAAAACATCTCTACG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 28 GAACGGGTACGAGATTCCGGGAGGTGACGAACGAACGTAGAGATGTTTTTGGTAACTTTG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 29 CGGAATCTCGTACCCGTTCCCAGAAAATGCTGGAAGGTACTTCCGGTGAAAACGGCGAAT 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 30 TTATTCGCCGTTTTCACCGG 20

__**Nanodrop with pGBR22**__



Figure 1: The first run of Nano drop to determine the concentration and purity of our pGBR22.



Figure 2: The second run of Nanodrop that confirmed our initial readings of the concentration and purity of the pGBR22.