Alyssa+K.


 * __Week of 12/2/13-12/6/13__**

DH5-Alpha cells of the pfFabG plasmid were midi-prepped and nanodropped for storage in the -20 degree Celsius freezer in confirmed plasmid storage. The results of the Chembridge library were also collected and the top 10 ligands were found.

Table 1. Top 10 ligands from the 306 ligand Chembridge library screened. Although these scores were higher than the ones from the control docking, they are still lower than the ideal range of 80-100 that we would be looking for an effective ligand to test in assays. The protein structure was not very good and a better crystal structure needs to be found or a homology model made before more screening can be done. __**Week o****f 11/25/13-11/29/13:**__ Thanksgiving Break!
 * __Week of 11/18/13-11/22/13__**

An enzyme assay protocol for pfFabG was carried out. This was an inital enzyme assay and was a check to make sure the protein was functional. A library screening of the pfGR target was also carried on the the 306 ligand chembridge library. The above shows the UV spectrum of only the buffer, water, and Acetyl CoA reagents.

The above graph shows only the buffer, water, and NADPH reagents. Although not shown in this figure, it should be noted that at 340 nm the absorbance is 0.112 which is the absorbance of NADPH.

Erroes in both of the above two graphs could include not allowing the reagents enough time to reach equilibrium before spectroscopy. The graph above includes all reagents with the NADPH and FabG enzyme being added in during the data collection.

The first peak shows the addition of NADPH. It should be noted that additional NADPH was added because with the cuvette already inserted it was difficult to determine if the small amount of reagent was added to the mix. The second peak shows the addition of pfFabG. In future enzyme assays, more enzyme should be added to bring down the absorbance more.

On Monday, protein characterization was done. The gel was run and then washed and stained and allowed to de-stain overnight. Elution one was concentrated and stored in snap-freeze and glycerol. The next day, the gel was taken out and found to have a large tear in it so it was not dried but a preliminary picture was taken.
 * __Week of 11/11/13-11/16/13__**

Control Docking rankings for pfGR.


 * __Week of 11/4/13-11/9/13__ **

The control docking was resubmitted because it failed to run, may have been booted. On Tuesday morning, small liquid cultures (10 X 4ml LB) were inoculated with pfFabG and incubated for 8 hours. The large-culture step was done that evening and incubated for 18 hours at room temperature. The incubated liquid culture was then spun down using the JA-10 rotor in two 500 ml cylindrical bottles on Wednesday afternoon. The samples were re suspended in lysis buffer and stored in the -80 degree Celsius freezer. On Friday, cell lysis was done by sonication and the sonicated samples were spun down, the supernatant was retained and then clarified by filtration. On Saturday morning, the Ni-NTA and His-tag purification protocol was followed out on the sample and both elutions were analyzed with Nanodrop spectroscopy. All samples were stored in the 4 degree Celsius freezer. Table 1. Starter culture added and OD readings of large culture before induction.



Good work Alyssa! Try to include more analysis of your procedures and results. -Suman 11/4/13
 * __Week of 10/28/13-11/1/13__**

The plates were master plated and liquid cultures were grown over night. The next day no growth was seen in the master plate or liquid culture, so the bacteria were not viable after the long incubation. Virtual screening was done and NADP and GSH were docked into the 1ONF crystal structure and a control docking was run. I switched to work on the FabG target with Melissa. lB media was made and autoclaved and a master plate and small liquid culture were made of the validated DH5-alpha colony to make more plasmid later for future transformations. The control docking job was also resubmitted because it got dropped the first time.



Positive Controls for pfGR Negative Controls for pfGR
 * **Compound ** || **IC50 ** || **Mol. Weight ** || **LogP ** || **H Donor ** || **H Acceptor ** ||
 * GSHoriginal || Original  || 307.3  || -4.5  || 6  || 8  ||
 * 573747pos1 || 40 nM  || 306.3 || 3.4  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">0  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">7  ||
 * <span style="font-family: Calibri,sans-serif; font-size: 11pt;">45484010pos2 || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">63 nM  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">221.1 || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">1.7  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">0  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">6  ||
 * <span style="font-family: Calibri,sans-serif; font-size: 11pt;">10352496pos3 || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">110 nM  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">190.1 || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">1.5  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">0  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">4  ||
 * <span style="font-family: Calibri,sans-serif; font-size: 11pt;">44626409pos4 || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">350 nM  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">302.2 || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">1.2  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">0  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">9  ||
 * <span style="font-family: Calibri,sans-serif; font-size: 11pt;">45484014pos5 || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">400 nM  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">302.2 || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">1.2  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">0  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">9  ||
 * <span style="font-family: Calibri,sans-serif; font-size: 11pt;">25270070pos6 || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">480 nM  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">314.1  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">1.6  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">0  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">9  ||
 * <span style="font-family: Calibri,sans-serif; font-size: 11pt;">5325382pos7 || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">630 nM  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">206.1 || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">2.1  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">1  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">5  ||
 * <span style="font-family: Calibri,sans-serif; font-size: 11pt;">651069pos8 || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">4500 nM  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">289.3  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">1.1  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">1  || <span style="font-family: Calibri,sans-serif; font-size: 11pt;">4  ||
 * Compound || Mol. Weight || LogP || H-donor || H-acceptor ||
 * 2244 (aspirin) || 180.1 || 1.2 || 1 || 4 ||
 * 40648952neg1 || 290.3 || .3 || 3 || 4 ||
 * 3082400neg2 || 303.3 || -8.2 || 5 || 9 ||
 * 440634neg3 || 303.3 || -8.2 || 5 || 9 ||
 * 1598670neg4 || 290.2 || -1.8 || 4 || 4 ||

The gel extraction protocol was performed on the below gel. The concentration on this gel was very low, so another secondary PCR was done, as well as a PCR squared. 300 microliters of the PCR squared sample was run on a gel and gel extracted. This DNA sample was used as the PCR insert for transformation along with the pNIC vector cut on October 16th. Transformation was done and plates were checked the next afternoon with no growoth. There was no growth the next morning as well. A day and a half later, colonies were present and stored in the four degree fridge for the master plate protocol. Gel extraction of the PCR squared sample from last week was done. The concentration of this gel extraction was low and the previous cut pNic vector was not digested fully, so more of each was made. pNIC digestion was successful, but two PCR squared protocols resulted in primer dimers. The primers were rediluted from the original stocks to resolve this problem. The gel was hen cut and stored for gel extraction.
 * __Week of 10/21/13-10/26/13__**
 * __Week of 10/14/13-10/19/13__**
 * Nice work on keeping the gel extraction data posted. Good captions and analysis. Try to include your gene size on the gel captions. Do you have any virtual data? Thank you. -Max 10/21/13

This gel was extracted despite primer dimers, because they would not be included in the gel extraction protocol anyways. The concentration on the above gel extraction was very low, so more sample needed to be made and gel extracted again.

The below two gels are from two separate PCR squared samples that were both run. This gel resulted in primer dimers which with very little existence of the target gene present. The pNIC in this gel is shown to be digested more fully but the PCR sample is not the correct length of 1500 bp. This is the Nanodrop of the cut pNIC. Purity looks good. Another PCR squared was run with the same primers but newly diluted dNTP, so the problem was reasoned to be the primers which were diluted again in the next gel. This was PCR squared with the new diluted primers, some dimers still present, but will be cut out anyways so only the target gene will be in the gel extraction.


 * __Week of 10/7/13-10/11/13__**

Another secondary PCR was done from the Primary max sample. The next day the secondary PCR sample and a PCR squared sample was ran to confirm them before moving on to gel extraction. Lane 1: 1 kb ladder Lane 2: Secondary PCR at 63 <span style="background-color: #ffffff; color: #444444; font-family: arial,sans-serif; font-size: small; line-height: 1.5;">°C annealing temp Lane 3: PCR squared sample

Both sample are good for further use in cloning. The secondary PCR sample will be saved if more PCR squared sample needs to be made in the future and gel extraction will be done on the remaining PCR squared sample.


 * __Week of 9/30/13-10/4/13__**


 * Nice captions and brief analyses. Keep up the good work. -Max 10/07/2013
 * Clone results came back again with no significant match for colony one. Another gel extraction was done along with cutting of more pNIC-Bsa4 vector. The concentration on the gel extraction was very low, so another secondary PCR and PCR squared were made in preparation for another gel extraction protocol.**




 * Lane 1: 1 Kb ladder**
 * Lane 2: Primary PCR**
 * Lane 3: Secondary PCR (@ 63 degree annealing temp)**
 * Lane 4: PCR squared **
 * Lane 5: nothing**
 * Lane 6: Cut pNIC**


 * The primary looked good, but secondary and PCR squared were not bright or definite enough. Another run of secondary and PCR squared will have to be done to produce a good enough sample for gel extraction and then go into cloning again.**

code NNNNNNNNNNNNNGNGGNGGNTCTGGNGGTATGGCGGCTGCAGCCATGGATGAAATAACAGTAAAGGTGGATACGGATCC GAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAG CCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTG AGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCG GGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTT TCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGC ACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTG ACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTT TGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTA ACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAA TACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCAT ATCNNGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGNAAGGAGAAAACTCACCGAGGCAGTTCCATAGG ATGGCAAGATCCTGGTATCGGTCTGCNATTCCNACTCGTCCAACATCNATACAACCTATTAATTTCCCNCGTCAAAAATN ANGTNATCAAGTGAGAAATCACCATGAGTGACNACTGNATNNNNNANNANGNAAANGTTTATGCATTTCTTTNNNNANNN NNANNNNNNNNNTACNCNTCNNCATNAAANTNNNTCGCATNANNANCNTNNTTCNTNNNNATGNCCNNNNCNNNNCNNAN NNNNCNATNNCNNNNNNNNNNNNNNNANNNNNANCNANNNNNNNNNNNNGAANNNNNNNNNNNNNNTNNANNAAANN code
 * Sequencing results for Colony 1 with no significant similarities to our gene.**
 * Concentrations of this DNA sample are too low. Secondary and PCR squared will be done again to create more sample for another gel extraction.**

__Week of 9/23/13-9/28/13__
 * Sequence results for colony 1 came back and after a pairwise alignment with the PfGR codon optimized gene sequence, it was found to have no similarity. Closer analysis of the sequence results shows that many of the nucleotides were not able to be determined. The cohesive end generation and transformation process was repeated with the new gel extraction sample from last week. Two more sequencing samples from colony 1 were sent (one with forward primer, one with reverse primer) to see if better results could be received. After 2 days in the incubator, no colonies were present on plate A or B from this week's round of cloning.**


 * For next week: Because the 2 V: 8 I ratio has not been successful, a higher ratio (possibly 1 V: 9 I) will be used for the next round of cloning.**

1ONF**: Plasmodium falciparum glutathione reductase; FAD only (cofactor, not localized in same vicinity as NAD)** 3SQP**: Human homology model with antimalarial agent bound; 40% identity, align well in PyMol** 1GEU**: E.coli; has FAD and NAD; *use this to define NADPH binding site*** 1GET**: E.coli; FAD and NADPH; NADPH and FAD in another chain when aligned** 4G0L**: E.coli glutathione-hydroquinone reductase; glutathione is bound; not aligned well** 3R3E**: E.coli glutathione transferase;glutathione bound; not aligned well** 2HQM**: Yeast glutathione reductase; glutathione bound and matches up well, but glutathione doesn't seem to be in active site** 2GRT**: Human glutathione reductase, aligns well, NAD and Fad line up, FAD lines up with GEU, GDS not close enough** 1DNC**: Human Glutathione Reductase, aligns well, FAD aligns with 1ONF and 1GEU, GSH not close enough to active site** 1GRA**: Glutathione Reductase, aligns well, FAD aligns, NDP does not, GSH not close enough** 1GRE**: aligns well, FAD aligns, GSH not close to active site, 16 polar contacts** 1GSN: **aligns well, FAD aligns, GSH is close to active site, 8 polar contacts** 3DK4: **aligns well, FAD aligns, GSH similar to 1GSN (accept has 2 ligands), 18 polar contacts** 3DK8: **aligns well, FAD aligns, same GSH binding as 3DK8, 15 polar contacts**
 * Virtual Screening Set-up**


 * To-Do: **
 * Look at mitochondrial GRs**
 * Dock NADPH into 1ONF from 1GEU**
 * Find protein for glutathione docking**

code NNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNANNNNNGGNNNNNNNTNGGTGANTANCACNNAGNNNGGNNNNNNNNNNN NNNTNTGNNNANNNTNTNGNNNNNNNTTCNNTANNCNNNNCANANNNTNAGNTCNNANNGNNNNAANNNNNNNNNGANNN NNNTNNNTNCNNTNCCCGNNNANCNTNCNNNNNNANTCCCTTCGNNGCTCCANACAGNTCTNNANGGATTTTTTCCNNAA ATGANNACATNNGANTAGAAAGGANAATGNGAGNGNCCCCGCAANGGCNCNNTCNNCCCCCCCGGNNTGNNGGCNNNNNN NNNTNCNNNCNNNNTCNCNANCNNCTNNNNCNATCCCNTTNNNCNNTNTNCCNTNCTTNCTCNCCNNGANCGTNNGCNTN NNNGNNCAANCNNTNNAGCNNGNNCTCCCNNNCNGGNNNNGATTNNGNNCATNANNNTNACTCNCCCCCTACAAAGGTGA GNTNGNNNNNNGTTNNAGANNNGCTGCGNTNNCNNCNAATTANNGNTTTTTTNCNNNNGNNNNCNNTAGNTNNTNTNNNN NNTNNCNANNCTTGGNNTTNAGTNGANNNNNNTTNNNTCNNATCNAACNCNTNCTNNNNN code Week 3 & 4 9-Sep - 22-Sep __- use stephanie to make back up materials!__
 * Sequence Results from Colony 1**
 * Alyssa - ok - you need to keep pushing the cloning forward. We want to get you onto protein work soon. Get set up to try multiple rounds of cloning and always make extra materials (PCRsquared and pNIC-bsa4 - so that you don't have to go back 2 steps each time) - Dr. B 100113**

__Week of 9/16/13-9/20/13__
 * On Monday, the first day of cloning was done. DH5-Alpha cells were transformed with the pNIC-Bsa4 which was inserted with our PCR insert. Tube A was done with 2µl of accepting vector with 8µl of PCR insert. Tube B had 1µl of accepting vector with 7µl of PCR insert. The transformations were plated and incubated overnight. Plate A had only one colony after one night of incubation and Plate B had none, so both plates were incubated for another night. After two nights of incubation, only one colony was able to be collected (the same one from plate A). Colony 1 from plate A was incubated over night in an LB culture and spun down the next morning. A sample for colony 1 was prepared and sent off to sequencing. Another gel extraction was done so more DNA sample would be ready for cloning the next week. **


 * The purity and concentration of this DNA sample from the gel extraction on 9/20/13 are both within a good range. However, the contamination reading (260/230) is fairly low. It should be **


 * around 2.1. This could possibly be due to residues added to the sample from the gel extraction kit. **



__Week of 09/9/13-9/13/13__ __Week of 9/2/13-9/6/13__
 * PCR squared was done again on secondary sample Hm. All samples were then run on an agarose gel and gel extraction was done. The DNA sample was split into 10 collection tubes after gel extraction. PCR cleanup on the 10 samples was done to combine them into 2 more highly concentrated samples. These samples were however fairly low. Later another gel extraction was done on the remaining 3 PCR squared samples. Then another PCR cleanup was done with the three samples to get one concentrated samples of DNA. Two more PCR squared samples were made in case more DNA sample was needed.**
 * After receiving this suitable concentration of DNA sample (that we know is all our gene because of gel extraction) we will move on into cutting PNic-BSA4 and cohesive end generation and transformation next week.**
 * Sequencing results for clones were analyzed. Sequence results both forward and reverse were no more than 30-40 bp long. A smaller DNA piece must have attached and cloned instead of the target gene. To solve this problem, another PCR gel will be run and then gel extracted.**

__Week 9__
 * Mini-prep was done on the 16 samples and they were prepped for sequencing.**
 * Samples C1, C2, C3, C4, C5, and C8 were chosen based on purity to be sequenced with both forward primers and reverse primers, so in total 12 samples were initially sequenced.**

__Week 8__
 * PCR squared was done on the Hm sample with and annealing temperature of 63°C. pNIC-Bsa4 was cut with BSAI-HF and cohesive end generation was done on both the PCR insert and the accepting pNIC vector. Transformation was completed and the first set of plates did not grow any colonies. The next day another transformation was done with the previous vector and PCR insert that had cohesive end generation done and with new samples with cohesive end generation. The plates with the second cohesive end generation done to them produced colonies and eight colonies from each plate (plate C and D) so a total of 16 samples were used for the two master plates and the small cultures. The cultures were spun down and put in the freezer for mini-prep next week. **

__Week 7__
 * Sequencing results came back from the pNIC-Bsa4 plasmid made last week and were analyzed. Secondary PCR overlap of the oligos for PfGR was redone with a range of temperatures to find the optimal annealing temperature with both first and last oligos and tail primers. Another secondary PCR with a higher range of temperatures were done the next day. The next day two gradients were done on a secondary PCR with a new oligo mix made with tris base and a new primary. Successful secondary PCR was done by doing 30 second annealing and extension times for both the primary and secondary. **
 * Lane 1: 1 Kb ladder**
 * Lane 2: Max Primary**
 * Lane 3: Hm (63°C)**
 * Lane 4: Im (63.6°C)**
 * Lane 5: Jm (64.6°C)**
 * Lane 6: Km (66.2°C)**
 * Lane 7: Lm (68.1°C)**
 * Lane 8: Mm (69.7°C)**
 * Lane 9: Nm (70.6°C)**
 * Lane 10: Om (71.0°C)**
 * Lane 1: 1 Kb ladder **
 * Lane 2: primary **
 * Lane 3: PCR squared **
 * Lane 4: B° (62.7 °C) **
 * Lane 5: C° (63.2°C) **
 * Lane 6: D° (63.9°C) **
 * Lane 7: E° (64.7°C) **
 * Lane 8: F° (65.4°C) **
 * Lane 9: G° (65.8°C) **
 * Lane 10: nothing **
 * Lane 1: 1 Kb ladder**
 * Lane 2: I (62°C)**
 * Lane 3: J (61.8°C)**
 * Lane 4: K (61.4°C)**
 * Lane 5: L (60.6°C)**
 * Lane 6: M (59.6°C)**
 * Lane 7: N (58.8°C)**
 * Lane 8: O (58.3°C)**
 * Lane 9: P (58.0°C)**
 * Lane 10: nothing**
 * Lane 1: 1 Kb ladder **
 * Lane 2: Q- (72°C) **
 * Lane 3: R- (71.8°C) **
 * Lane 4: S- (71.4°C) **
 * Lane 5: T- (70.6°C) **
 * Lane 6: U- (69.6°C) **
 * Lane 7: V- (68.8°C) **
 * Lane 8: W- (68.3°C) **
 * Lane 9: X- (68.0°C) **
 * Lane 10: Primary **


 * Lane 1: 1 Kb ladder**
 * Lane 2: Sample S (65°C)**
 * Lane 3: Sample T (65.2°C)**
 * Lane 4: Sample U (65.7°C)**
 * Lane 5: Sample V (66.4°C)**
 * Lane 6: Sample W (67.2°C)**
 * Lane 7: Sample X (67.9°C)**
 * Lane 8: Sample Y (68.3°C)**
 * Lane 9: Sample Z (68.5°C)**
 * Lane 10: Sample A^2 (PCR squared with samples D, E, F, G, from below)**


 * Gene is ~1500 bp**
 * Lane 1: Sample A (62.5 ** ° ** C) **
 * Lane 2: Sample B (62.7 °C) **
 * Lane 3: Sample C (63.2 °C)**
 * Lane 4: Sample D (63.9 °C)**
 * Lane 5: Sample E (64.7 °C)**
 * Lane 6: Sample F (65.4 °C)**
 * Lane 7: Sample G (65.8 °C)**
 * Lane 8: Sample H (66 °C)**
 * Lane 9: 1 Kb ladder**




 * DNA sequencing results from pNIC-Bsa4 plasmid**
 * NNNNNNNNNGTTNACTTTAGANGAGANATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGA GAACCTGTACTTCCAATCCATGGAGACCGACGTCCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTAT GATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGA AACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATA GACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGG CAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCG TAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACG ATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGC GTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAA TCCCTGAACAGCAAAAAAATGAAAAATATAAAGTTCCTGAGTTCGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAA GGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACACTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTT TGCATTAGCCGGANATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGNGAAACTTCTATTG ACAGCTNGGAAAACGCTGGNCGCGTCNTTAAAGACAGCGACNAANTCGATGCANTGATNCTATCCTAAAAGACCNAACNN ANANNGNCNGNTCAGCCNCNTTTNCNTCTGANNGAAAATCNNNNTCTACNCTGATTCNNCNGTAANNNTTACNGNAANNA NNNCTGNNACTGNNNNANNNACGNNNNNNNNNTNNNNNCNCTTNAANNTCNNCGNNGNNNNGNATNNNANNNNNNTNNAN GNNNNNNNNANNNNNNNNNNNCNNNNNNCNANNNGNANNNNNNNNNNNNGGNNNNNANNNNNNNNNNNNTNNNNNNNNN**

__Week 6__ __Week 5__ __Week 4__ __Week 3__
 * Pairwise Alignment with pNIC-Bsa4**
 * The secondary PCR overlap protocol was finished and the samples were prepped and run on an agarose gel the next day for analysis. FPLC of the PfDXR protein was done for further purification of this protein. VS4 was redone again, but this time CONECTs between the metals and other residues were removed. Midi prep of the pNIC-Bsa4 protein was also done and the sample's concentration was determined by Nanodrop and was sent to sequencing. Two more secondary PCRs were done, one with first and last oligos, the seconds with tail primers. Both of these were also run on a gel. The protein samples recovered from the FPLC of PfDXR were spun down and filtered and then analyzed with Nanodrop. The proteins were then stored by both flash freeze and glycerol methods.**
 * The below gel shows the secondary PCR of PfGR done with an annealing temperature of 64 degrees Celsius, the above is at 63 degrees Celsius. The next attempt will be done at 64 degrees since the below image shows some amplification at the appropriate bp length (about 1500 bp).**
 * Primer design of the tail primers for our specific targets were done and put on the wish-list for ordering. Then primers for our targets were diluted and stored to run a gel on later. Another culture of pNIC-Bsa4 was grown overnight and spun down and saved in the -20 freezer for midi prep to be done to it later. An oligo mix was made from the 38 primers for the 3D7 glutathione reductase target. From the oligo mix a PCR overlap protocol was done and an agarose gel was run from the primary and secondary samples. The primary sample could be made out on the gel. but not the secondary sample so another primary and secondary PCR overlap was made, as well as another secondary sample from the initial primary sample.**
 * Analysis of DNA sequencing of pGBR22 was done in the computer lab in which the forward and reverse primers were determined, as well as the location of the start and stop codon of the specified plasmid and the vector backbone. SDS-Page protein gels were prepared Tuesday for later use on our purified protein. pfDXR protein samples from last week's protein expression were sonicated and then purified. Both elution samples were then analyzed on the Nanodrop spectrometer. The next morning the samples taken from protein purification of pfDXR were run on the SDS-Page protein gel made earlier in the week and was washed and stained. An agarose gel from the pLIC PCR that was redone last week was redone. After washing overnight, the SDS-Page gel was dried for analysis. Restriction enzyme digest was also done and an agarose gel was done for analysis. **
 * The PCR of the GFP sample done last week was run on a gel and imaged for analysis. The results from the DNA sequencing of the midi-prepped pNIC-Bsa4 sample were also returned and analyzed. Then two small overnight cultures of BL21(DE3) with FABV plasmid (100 ml LB each) were prepared and left in the shaking incubator overnight. A larger LB flask (500 ml LB) was also prepared to transfer the small culture over the next morning. The next morning the FABV bacteria did not have any growth so protein expression was continued with the pfDXR culture. 30 ml of the pfDXR culture was transferred to a larger culture was checked every 30 minutes until it reach an OD 600 of 0.500 (reached 0.995) and then left in the shaking incubator for approximately 3.5 hours. This culture was then spun down and re-suspended in buffer to be stored in the -80 degrees Celsius freezer. PCR of pNIC-Bsa4 plasmid was done with pLIC primers and the samples were run on an agarose gel and imaged for analysis.**



code NNNNNNNNNNNNNTTTAGNNGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGA ACCTGTACTTCCAATCCATGGAGACCGACGTCCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGA TATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAA CAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGA CCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCA AGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTA ACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTNACACAGTACATAAAAAAGGAGACATGAACGAT GAACNTCNNAAAGTTTGCNAATTTGNNNCNNTANNAANCNTTACTACCNCACTGCNGGCANGANGTTCTACTNAAGNNNN NNCNAANNNNNNCACCNNNCNNNNNANNNNGAANCATNNNNGNNTNCCCATNNNNNNNCNNTGANNNNCTGCTANNNNCT GATNAGCNNNANNATNAANAATNTNNAGANNNNTGTTTTTTATTNTANTNNNNNNNNNNNNNNNNCGANCTCATTGCGCC NNNCTGNCTNNNNTCTTTNNNTCNNNNNGAANNNCNNCA code
 * DNA Sequencing results for pNIC-Bsa4**
 * Blast Results for above sequence**
 * The above confirmed the indentity of the pNIC-Bsa4 plasmid and it was transfered to the plasmid box.**

__Week 2 - A - great work and great page! -- Dr. B__ __Week 1__
 * Midi Prep was done on the pNic-BSA4 plasmid sample that was made from the overnight culture last week. Agarose gels of last week's PCR samples were made and analyzed. The first midi prep did not produce a successful sample, so a second culture was made and spun down for a second midi prep to be done. The sample collected from the midi prep was then analyzed using the Nanodrop spectrometer and found to have a concentration of 19.6 ng/ul. A second PCR was done using VDS 1 and 2 primers as well as M13 forward and reverse primers on the GFP plasmid. **
 * Primer T7 was diluted to a 10uM concentration. DNA sequencing of pGBR22 plasmid was also done and the results came back with an exact match for the chromoprotein. E.coli cells were transformed with pNic-BSa4 plasmid, and overnight culture was made, and that culture was then spun down and stored for the midi prep protocol. PCR was also done to the same pGBR22 plasmid and the samples were stored in the freezer. **

Transformation






 * Transformation Efficiency Calculations**


 * Plate A: 245**


 * Plate B: 30.4**

DNA Sequencing
 * Plate C: 3.32**


 * pGBR22 plasmid **


 * NNNNNNNNNNNNNGGGCGATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGGGATTTTAGTGATGGTGATGG **
 * TGATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTAC **
 * ATCCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACA **
 * AATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCA **
 * GTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATT **
 * GTAGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCT **
 * CCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCG **
 * TATTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTC **
 * CCCCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGT **
 * AGGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCG **
 * ACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCA **
 * TGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAA **
 * AGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTNNNAAANCTGTC **
 * NTGNCAGCTGCATTAATGAATCGGCNACNNNCGGGGNANNNGNNGTTTGCGTANTGNNNCTCNTCCGNTNCNCGCTCANT **
 * GANTCNNNNNNCTCNNNCNNNCGGCNNCNGNNNNNNNGNNTCANCNNNNNNNNNNGNNNGNNNNNNNNNTNNNCCNNNNN **
 * NNNGNNNNNACNNNGAANANNNNNNNNNNNNNNCNAANGCNNNNNNNNNAAAGNCNNNNNNNNNNNNNNNNNNTNNNNNN **


 * Blast Results (top match)**

Quantifying DNA**