Ashlee+W.

WEEK THIRTEEN (11/17/13-11/22/13) AND WEEK FOURTEEN (12/2/13-12/6/13): Before Thanksgiving break, my partner and I, Marianna U., completed the cloning protocol, and unfortunately, we did not find any positive clones. Because of this, we had to move on to the surrogate protein YopH enzyme. We then moved on in our research, using YopH for Protein Expression, Protein Purification, and Protein Characterization. We started option B at 8 PM. Purification was performed for YopH. According to the SDS Page Gel, the results appeared accurate according to Lane 7 that had Elution 1 and Lane 8 that had Elution 2. Both lanes had distinct bands.

__Figure 1:__ YopH enzyme absorbance of wavelength versus absorbance until the OD600 reaches 0.500 in order to add IPTG for the induction step of expression of T7 polymerase.

__Figure 2:__ Absorbance measurements from LoggerPro which are read at a measurement 600 nm to determine the OD600.

__Figure 3:__ Absorbance measurement from Nanodrop, which had a concentration 3.3 ng/uL for Elution 1.

__Figure 4:__ Elution 1 and Elution 2 after the purification of YopH. __Figure 5:__ Protein Characterization Gel. Lane 1 consisted of the Protein ladder. Lanes 2 and 3 consisted of the cell lysate after induction. Lane 4 consisted of the soluble fraction. Lane 5 consisted of the flow through. Lane 6 consisted of the wash solution. Lane 7 consisted of Elution 1. Lane 8 consisted of Elution 2.

WEEK TWELVE (11/11/13-11/15/13) AND WEEK ELEVEN (11/4/13-11/8/13): After Miniprepping our eight samples, we Nanodropped each sample to determine which two samples contained the highest concentrations, which would then be sent to the DNA sequencing facility (MBB 1.426). The concentrations for each sample included: Sample 1- 39.5 ng/uL; Sample 2- 19.5 ng/uL; Sample 3- 12.8 ng/uL; Sample 4- 43.0 ng/uL; Sample 5- 43.4 ng/uL; Sample 6- 16.1 ng/uL; Sample 7-29.0 ng/uL; Sample 8- 12.5 ng/uL. The two samples with the highest concentrations were Sample 4 and Sample 5. Since this was the case, 7 ul of each sample was micropipeted into a 1.7 ul microcentrifuge tube and sent off to the DNA sequencing facility. As we wait for our results, we will move on to work in virtual screening. We will continue to maintain accurate and precise work, along with ensuring sterile techniques in the wet lab, so we can progress in our research.

__Figure 1:__ 2 ul of T4-treated Accepting Vector mixed with 4 ul of the T4-treated Insert plated on a plate treated with Kanamycin (Tube A) left to grow in the media for one day.

__Figure 2:__ 2 ul of the T4-treated Accepting Vector mixed with 8 ul of the T4-treated Insert plated on a plate treated with the antibiotic Kanamycin (Tube B) left to grow in the media for one day.

__Figure 3:__ Master Plate after selecting eight colonies and incubating overnight plated on a Kanamycin treated plate.

__Figure 4:__ After miniprepping, this Nanodrop showed one of our highest concentrations, 43.0 ng/uL (Sample 4), amongst the eight samples Nanodropped.

__Figure 5:__ After miniprepping, this Nanodrop showed one of our highest concentrations, 43.3 ng/uL (Sample 5), amongst the eight samples.

WEEK NINE (10/21/13-10/25/13) AND WEEK TEN (10/28/13-11/1/13): After PCR squared, Marianna and I Nanodropped in order to determine the concentrations of our samples that were successful. From there, the most successful samples with the highest concentrations (Sample A: 19.8 ng/uL and Sample G: 19.1 ng/uL ) underwent PCR clean up in order to verify if pNIC-Bsa4 cut correctly. Although our results in PCR clean up were successful, the bands in Lane 2 were very faint, so we had to just assume that since Sample A in Lane 1 was successful, Lane 2 was also successful (Figure 3). After PCR cleanup, we Nanodropped to determine the concentration. The concentration was 19.6 ng/uL, which isn't the best concentration, but we will still move on to the next steps in our research. Then, we continued on to Cohesive End Generation on PCR inserts, as well as Cohesive End Generation of Accepting Vectors, but we do not have any images to display, as we are still waiting on results.We will continue to maintain sterile techniques, along with accuracy and precision while completing our next steps in our research.

__Figure 1:__ Nanodrop after successful attempt at PCR squared with Sample A (run on a 10°C temperature gradient at 63°C). The concentration of this sample is 19.8 ng/uL.

__Figure 2:__ Nanodrop after successful attempt at PCR squared with Sample G (run on a 10°C temperature gradient at 54°C). The concentration of this sample is 19.1 ng/uL.

__Figure 3:__ PCR Clean up. Lane 1 consists of 5 ul of the 500 base pair DNA ladder. Lane 2 consists of 3 ul of Sample A ( run on a 10°C temperature gradient at 63°C) + 5 ul of blue juice (8 ul total). Lane 3 consists of 3 ul of Sample G (run on a 10°C temperature gradient at 54°C) + 5 ul of blue juice (8 ul total).

__Figure 4:__ Nanodrop of PCR clean up. The concentration is 19.6 ng/uL.

__WEEK EIGHT (10/14/13-10/18/13):__ Great work in the wet lab. Nice captions and analysis. Do you have any virtual data? Thank you. -Max 10/21/13 After successfully completing Primary PCR, Marianna U. and I moved on to Secondary PCR, which the samples A (run at 63 degrees Celsius), G (run at 54 degrees Celsius), and H (run at 53 degrees Celsius) were the brightest and all showed very promising results. We used a temperature gradient of 10 degrees Celsius, and now we know which temperatures to use if we were to ever re-do Primary PCR. We will continue to use sterile techniques in lab, as well as make sure that we are using the correct concentrations, temperatures, and waiting the correct amount of time in order to ensure accuracy and precision in our research. We will move on to PCR clean up on Tuesday.

__Figure 1:__ Secondary PCR attempt (first try) by Ashlee W. and Marianna U. The Primary PCR samples used for Secondary PCR were Sample A (63.0 degrees Celsius), Sample G (54.0 degrees Celsius), and Sample H (53.0 degrees Celsius). Lane 1 contains 5 ul of the 100 base pair DNA ladder, Lane 3 contains Sample A from Primary PCR, Lane 4 contains Sample G from Primary PCR, Lane 5 contains Sample H from Primary PCR, and Lane 7 contains 5 ul of the 100 base pair DNA ladder. Lanes 4, 5, and 6 contain 15 ul of one of the Primary PCR samples + 5 ul of blue juice (20 ul total). Our samples amplified successfully.

__Figure 2:__ PCR squared by Ashlee W. and Marianna U. Lane 1 was empty, Lane 2 contained 5 ul of the 100 base pair DNA ladder, Lane 3 contained 15 ul of Sample A (run at 63 degrees Celsius) + 5 ul of blue juice (20 ul total), Lane 4 contained 15 ul of Sample G (run at 54 degrees Celsius) + 5 ul of blue juice (20 ul total), Lane 5 contained 5 ul of the 100 base pair DNA ladder, and Lane 6 was empty. Although the 100 base pair did not completely and successfully run down the gel, Sample A and Sample H successfully amplified during PCR squared.

__ WEEK SEVEN (10/7/13-10/11/13): __

After many failed attempts at Primary PCR, we decided to not only try to run our samples on a temperature gradient 10 degrees Celsius, but we also tried to change the amount of dNTP used (2 ul of the 10 mM stock solution, rather than dilute it), and we ran Primary PCR twice, having 16 samples to run on a gel. We used a temperature gradient of 10 degrees Celsius in order to test a +/- 5 degrees Celsius gradient f the original temperature mentioned in the protocol of 58 degrees Celsius. This temperature consisted of temperatures between 53 degrees Celsius and 63 degrees Celsius, including: Sample A: 63.0 degrees Celsius, Sample B: 62.5 degrees Celsius, Sample C: 61.3 degrees Celsius, Sample D: 59.4 degrees Celsius, Sample E: 56.9 degrees Celsius, Sample F: 55.0 degrees Celsius, Sample G: 53.7 degrees Celsius, and Sample H: 53.0 degrees Celsius. In both Figure 1 and Figure 2, our 100 base pair DNA ladder did not run fully along the lane, which is odd because it should since it is only a 100 base pair DNA ladder. This could have resulted due to an issue occurring in our our gel preparation, such as using an incorrect amount of Agarose or 1X TAE Buffer. In both Figure 1 and Figure 2, all of our samples amplified, but the samples in Figure 1 were not as bright and promising as the samples in Figure 2. In Figure 2, Lane 2 (run at 63.0 degrees Celsius), Lane 8 (run at 53.7 degrees Celsius), and Lane 9 (run at 53.0 degrees Celsius), show very promising results, and these are the samples we will use to carry on to Secondary PCR, as the aligned with the 100 base pair DNA ladder in Lane 1 (Figure 2). Primary PCR consisted of taking random oligos to combine and make double stranded DNA that won't organize completely, resulting in a smear on the gel. Because we have successfully achieved amplification in our samples, we will now move on to Secondary PCR, where the fragments produced by Primary PCR will form bands based on the size of the DNA base pairs. We will then move on to PCR squared, where the same size bands will be overexposed, producing a great amount. We will continue to pay attention to using sterile conditions in the wet lab work space, precise and accurate concentrations, and devotion to precision and accuracy in the hopes of moving on and making progress in our research.

__Figure 1:__ Primary PCR attempt with a temperature gradient of 10 degrees Celsius. Lane 1 contains 5 ul of the 100 base pair DNA ladder. Lanes 2-9 contain 15 ul of each Primary PCR sample at a specific temperature + 5 ul of blue juice (20 ul total). The temperatures used were: Lane 2/Sample A: 63.0 degrees Celsius, Lane 3/Sample B: 62.5 degrees Celsius, Lane 4/Sample D: 61.3 degrees Celsius, Lane 5/Sample E: 59.4 degrees Celsius, Lane 6/Sample F: 55.0 degrees Celsius, Lane 7/Sample F: 55.0 degrees Celsius, Lane 8/Sample G: 53.7 degrees Celsius, Lane 9/Sample H: 53.0 degrees Celsius, and Lane 10 was empty. Although the gel did result in amplification in all samples run across the 10 degrees Celsius temperature gradient (53 degrees Celsius- 63 degrees Celsius. Original temperature in protocol: 58 degrees Celsius), the samples resulted in not being as bright as our successful attempt (Figure 2).

Figure 2: Primary PCR attempt with a temperature gradient of 10 degrees Celsius (53 degrees Celsius-63 degrees Celsius; Original temperature in protocol: 58 degrees Celsius). Lane 1 contains 5 ul of the 100 base pair DNA ladder. Lanes 2-9 contain 15 ul of each Primary PCR sample run at a specific temperature along the 10 degrees Celsius temperature gradient + 5 ul of blue juice (20 ul total). The temperatures used were: Lane 2/Sample A: 63.0 degrees Celsius, Lane 3/Sample B: 62.5 degrees Celsius, Lane 4/Sample D: 61.3 degrees Celsius, Lane 5/Sample E: 59.4 degrees Celsius, Lane 6/Sample F: 55.0 degrees Celsius, Lane 7/Sample F: 55.0 degrees Celsius, Lane 8/Sample G: 53.7 degrees Celsius, and Lane 9/Sample H: 53.0 degrees Celsius. Lane 10 was empty. Although the 100 base pair DNA ladder failed to spread across the entire Lane 1, our samples amplified; showing very bright smears for lanes 2, 8, and, which will be used for Secondary PCR.

__ WEEK SIX (9/30/13-10/04/13): __

After constant failed attempts at Primary PCR, my partner, Marianna U., and I decided to re-try Primary PCR using standard conditions while maintaining extremely sterile conditions and following the protocol intently. Unfortunately, our sample failed to amplify. For our next attempt, we are going to look into possibly purchasing new primers, using a different concentration of dNTP, or re-trying the temperature once more with more samples this time.

Figure 1: Primary PCR attempt by Ashlee W. and Marianna U. Lane 1 consists of 5 ul of the 100 base pair DNA ladder. Lane 2 consists of 5 ul of blue juice + 15 ul of our Primary PCR sample (20 ul total). The sample in Lane 2 failed to amplify.

__ WEEK FIVE (9/23/13-9/27/13): __ Due to issues with Primary PCR failing to amplify, we started off by using temperature gradient of 10 degrees Celsius, but only running samples with +/- 5 degrees Celsius, 53 degrees Celsius and 63 degrees Celsius, along this gradient. These two samples failed to amplify, so we decided to make a new oligo mix and run the samples along the entire 10 degrees Celsius temperature gradient: (Sample A: 63.0 degrees Celsius, Sample B: 62.5 degrees Celsius, Sample C: 61.3 degrees Celsius, Sample D: 59.4 degrees Celsius, Sample E: 56.9 degrees Celsius, Sample F: 55.0 degrees Celsius, Sample G: 53.7 degrees Celsius, and Sample H: 53.0 degrees Celsius). The gel was run with all 8 samples and a 100 base pair DNA ladder, but the DNA ladder and our samples failed to amplify, resulting in a blank gel for our first attempt (Figure 1). We then ran our samples on a new gel because we assumed that there was an issue with the machine, or the samples could have potentially ran in reverse. With running a new gel, we hoped that we would be able to pinpoint which temperature would result in amplification, so we could move on to secondary PCR and the rest our research. The samples also failed to amplify in our second attempt (Figure 2). In our next attempts, we will do more research on how to successfully complete Primary PCR, try the temperature gradient again, and also manipulate some more times, such as elongating the annealing temperature, so the primers have enough time to attach and prepare for DNA replication. We will also practice sterile techniques and make sure our concentrations of things, such as Agarose and our samples added to the wells, are precise and accurate.
 * Make sure to include week six data and crop your gels to make them smaller. Great analysis and captions. Thank you. -Max 10/07/2013

Figure 1: Primary PCR attempt using the full temperature gradient. Lanes 1 and 2 had 5 ul of the 100 base pair DNA ladder. Lanes 3-10 15 ul of each sample + 5 ul of blue juice (20 ul total). Each sample had a different temperature, including: Lane 3/Sample A: 63.0 degrees Celsius, Lane 4/Sample B: 62.5 degrees Celsius, Lane 5/Sample C: 61.3 degrees Celsius, Lane6/Sample D: 59.4 degrees Celsius, Lane7/Sample E: 56.9 degrees Celsius, Lane8/Sample F: 55.0 degrees Celsius, Lane 9/Sample G: 53.7 degrees Celsius, and Lane 10/Sample H: 53.0 degrees Celsius. Due to issues with the machine, everything, including the 5 ul of the 100 base pair DNA ladder, failed to amplify.

Figure 1: Primary PCR attempt using the full temperature gradient of 10 degrees Celsius. Each sample had a different temperature, including: Lane 1/Sample A: 63.0 degrees Celsius, Lane 2: 5 ul of 100 base pair DNA ladder, Lane 3/Sample B: 62.5 degrees Celsius, Lane 4/Sample C: 61.3 degrees Celsius, Lane 5/Sample D: 59.4 degrees Celsius, Lane 6/Sample E: 56.9 degrees Celsius, Lane 7/Sample F: 55.0 degrees Celsius, Lane 8/Sample G: 53.7 degrees Celsius, and Lane 9/Sample H: 53.0 degrees Celsius. Lanes 1 and 3-9 had 15 ul of the sample at a specific temperature + 5 ul of blue juice (20 ul total). Lane 2 had 5 ul of the 100 base pair DNA ladder. The gel run did not result in amplification.

__WEEKS THREE AND FOUR (9/9/13-9/13/13 and 9/16/13-9/20-13):__ Ashlee - ok, need more attempts at PCR here. We need to get y'alls cloning moving forward. Also, make some pNIC-Bsa4 for the cloning. - Dr. B 100113

After failed attempts at PCR using the standard temperature listed in the protocol, 58 degrees Celsius, we decided to use a temperature gradient of +/- 5 degrees Celsius, running the 53 degree Celsius sample and the 63 degrees Celsius sample in the gel. Because the samples both failed to amplify, we have decided to try run PCR along the entire concentration, which will result in 8 samples to run on the next gel. We will make sure to use extra sterile technique and complete each procedure with great accuracy and precision.

Figure 1: Primary PCR by Ashlee W. and Marianna U. Lane 1 consists of 5 ul of the 100 base pair DNA ladder. Lane 2 consists of 15 ul of the sample + 5 ul of blue juice (20 total) of our Primary PCR sample at the temperature listed in the protocol 58 degrees Celsius. The sample failed to amplify.

Figure 2: Primary PCR re-try. This time, two samples were run along a 10 degrees Celsius concentration gradient. Two samples with the temperatures, 53 degrees Celsius and 63 degrees Celsius, were used from this concentration gradient. Lane 1 consisted on 5 ul of the 100 base pair DNA ladder, and lane 2 had 5 ul of the 1 kb DNA ladder. Lanes 3 and 4 had 15 ul of either sample A or sample B + 5 ul of blue juice (20 ul total). Lanes 3 and 4, which contained the samples, failed to amplify.

__WEEK TWO (9/2/13-9/613):__ Ashlee - ok good work. Try more rounds of PCR and start to make your pNIC-Bsa4 in the meantime. - DR. B 090913

The Primary PCR failed to amplify, possibly for a variety of reasons, including: contamination, the agarose used to make the gel, or the PCR machine used. The next Primary PCR will consist of changes, such as making another oligo mix and testing a different temperature on each sample. One sample's temperature will be decreased by 5 degrees, and the other's will be increased by 5 degrees. Also, precautionary techniques, including maintaing a sterile environment, will be emphasized.

Figure 1: First attempt at Primary PCR by Ashlee W. and Marianna U. Lane 1 consists of 5 ul of the 100 base pairs DNA ladder. Lane 2 consists of 5 ul of the 1,000 base pairs 1 kb DNA ladder. Lane 3 consists of 20 ul (5 ul of blue juice and 15 ml of Primary PCR sample), but it failed to amplify.

__WEEK ONE (8/28/13-8/30/13):__ PyMol Refresher was completed in order to refresh the skills needed to successfully complete virtual screening.


 * __ Objective: __** to examine the three-dimensional structure of a new enzyme, in order to inhibit the growth of a parasite.


 * __ Materials: __** PyMol software, the proteins: 2H2Q, 3CL9, and 3HBB, the substrates: NAP, DU, MTX, and TMQ, and a Dell computer.

Figure 1: PyMol representation of the components of the protein 2H2Q. Chain A is colored red, and chain B is colored blue; both chains are displayed as sticks. Polar contacts between the protein and substrates, NAP and DU, are displayed as sticks and colored by elements on both chains. Hydrophobic, Ionic, and Polar residues are represented and colored black.



Figure 2: PyMol representation of theof the protein 3CL9, colored red and displayed as sticks.The substrate MTX is displayed as sticks and colored by element, with carbon as aqua. The substrate NAP is colored by element and shown as sticks, with carbon as green. The polar contacts between the inhibitor and the protein are colored black. The active site 5 angstroms from the ligand are highlighted in magenta.



Figure 3: PyMol representation of the components of the protein 1U72, displayed as sticks. The substrate NDP is displayed as sticks and colored by element, with carbon as green. The substrate MTX is displayed as sticks and colored by element, with carbon as aqua. The active site 5 angstroms from the ligand is highlighted in magenta.





Figure 4 and 5: PyMol representation of the proteins 1U72, active site colored aqua and displayed as sticks, and 3CLN, active site colored magenta and displayed as sticks, aligned. The RMS value is 1.126. The two substrates are superimposed within the active site; they share almost the same exact shape. If a new drug were added, it would be affected because the two substrates are super imposed and similar. The amino acid sequences appear to almost be identical because they are super imposed.

Figure 6: Blast pairwise comparison of the active sites of the proteins 1U72 and 3CL9. The two active sites scored very high in alignment.



Figure 7: PyMol representation of the protein 3HBB, displayed as sticks. Chain A is red, chain B is green, chain C is blue, and chain C is magenta. The substrate NAP is displayed as sticks and colored by element, with carbon is green. The substrate TMQ is displayed as sticks and colored by element, with carbon as aqua. The active site is highlighted in aqua, and the polar contacts are shown in the active site.

**__ Analysis: __** The substrates, NAP and MTX, of the two proteins, 1U72 and 3CL9, are super-imposed within the active site, meaning that they share almost the same shape and amino acid sequence. The two active sites of these scored very high in alignment, which proves that they have similar active site amino acid sequences.

**__ Conclusion: __** If a new drug were added to the substrates, NAP and MTX, if would be affectd because the two substrates are super-imposed and similar. The amio acid sequence, which scored very high in alignment are very close to being identical. When conducting this experiment, one should focus on clicking on the right part of the molecule in order to not make any further mistakes, throroughly read the protocol, and save images and sessions every few minutes, just in case a technical error occurred.

WEEK THREE: (6/17/13-6/21/13) _ __WEEK TWO: (6/10/13-6/14/13) - Good work Ashlee - Dr. B__

__Figure 5: VDS Ashlee W. 06/12/13 Run 1 of PCR of pGBR22.__ __Lane 1: Protein Ladder__ __Lane 2: Sample A__ __Lane 3: Sample B__ __Lane 4: Sample C__ __Lane 5: Sample D__

__Figure 5: VDS Ashlee W. and Karuna A. 06/14/13 Run 2 of PCR of pGBR22.__ __Lane 1: Protein Ladder__ __Lane 2: Sample A (Ashlee W.)__ __Lane 3: Sample B (Ashlee W.)__ __Lane 4: Sample C (Ashlee W.)__ __Lane 5: Sample D (Ashlee W.)__ __Lane 6: Protein Ladder__ __Lane 7: Sample A (Karuna A.)__ __Lane 8: Sample B (Karuna A.)__ __Lane 9: Sample C (Karuna A.)__ __Lane 10: Sample D (Karuna A.)__

__Figure 6: Absorbance reading at 260 nm of Sample 1 of pNic-BSA4 plasmid.__

__Figure 7: Absorbance reading at 260 nm of Sample 1 of pNic-BSA4 plasmid.__

_ WEEK ONE: (6/3/13-6/7/13) Figure 1: E. Coli (DH5-alpha) bacterial culture with 1 ng FrTUHP in pnic-Bsa4 after one day in 37 degrees Celsius incubator (10 colonies).

Figure 2: E. Coli (DH5-alpha) bacterial culture with 5 ng FrTUHP in pnic-Bsa4 after one day in 37 degrees Celsius incubator (3 colonies).

Figure 3: E.Coli (DH5-alpha) bacterial culture with 25 ng FrTUHP in pnic-Bsa4 after one day in 37 degrees Celsius incubator (0 colonies).

Since the plasmid used is a low count plasmid, the amount of colonies per plate resulted in being lower than expected. Because E.Coli competes for space, the higher the concentration of DNA plasmid, the less amount of E.Coli colonies per plate.

code NNNNNNNNNNNNNNCGANTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGGGATTTTAGTGATGGTGATGGTG ATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACAT CCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACAAA TAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGT GTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGT AGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCTCC CATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCGTA TTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCC CCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGTAG GTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCGAC CATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATG GTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG CCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTNNNAAANNTGTCNN GNCAGCTGCATTAATGATCGGCNANNNNCGGGGNNANGNNNNTNGCGTNTGGGNNCTCTTCNNNTCCTCNNTCANNGACT CNNNNNNNTCNNNCNNNNNNNNNNNNNNNNNGNNTCANNNNNNNNNANGNNNGNNNNNNNNNNNNNNNNNNNNNNNNNNN ANNNNNNNGNAANANNNNNNNNNNNNNNNNNNNNAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNNN NNN Figure 4: DNA sequencing-pGBR22. code