Editing Yeast Display Nanobody

Library recovery and expansion<br/>1. Place frozen aliquots of yeast nanobody library (NbLib) at 30 °C to thaw, such<br/>that recoverable yeast exceed library diversity at least five-fold. For the full<br/>naïve library, a total of 2.5×109 viable cells are required.

2. Recover yeast in 1 L of ‘Yglc4.5 –Trp’, shaking at 230 RPM, 30 °C, overnight. You<br/>may want to take a small sample of the library cells immediately after<br/>resuspension and plate on YPD agar to measure the diversity.

3. Expand to 3 L of medium and allow yeast to grow to stationary phase.<br/>Approximately 48 hours, OD is usually between 10 and 30.

4. Measure OD600 of yeast to calculate density (OD600 of 1 ≈ 1.5 × 107 yeast)

5. Spin down cultures at 3500×g for 5 minutes and resuspend in ‘Yglc4.5 –Trp’<br/>supplemented with 10% DMSO, such that final density is 1010 cells per mL.<br/>Aliquot to 2 mL cryovials and freeze in a cell freezing chamber at -80°C<br/>(Thermofisher Scientific cat# 5100-0001). Each vial should contain enough<br/>viable cells upon recovery to reconstitute a full library.

Induction of nanobody expression

Nanobody expression is under the control of the GAL1 promoter, so nanobodies are<br/>only produced on the cell surface when the yeast are grown in galactose-containing<br/>medium.

1. Induce nanobody expression of NbLib by dilution of ≥10 x library diversity into –<br/>Trp +galactose medium followed by shaking for 48 hours, 25 °C, 220 rpm. Use at<br/>least 5 × 109 yeast cells for the inoculum to ensure no clones are lost in<br/>passaging.

(We find best expression levels to be between 48 and 72 hours.)<br/>Note about sterility: It is essential to maintain a sterile environment throughout your<br/>selection. Unlike standard yeast work, your yeast cells will be passaged multiple times<br/>over the course of weeks, increasing the possibility of contamination. We recommend<br/>sterile filter tips and freshly sterilized media for every selection. Wipe down your<br/>hands, bench, and pipettes with ethanol often during the selection process. A sterile<br/>hood could be used as well, but usually a regular lab bench is fine so long as reasonable<br/>care is taken.

Nanobody expression test

Before any selection it’s a good idea to confirm that nanobody expression was<br/>effectively induced. We check this with a quick analytical flow cytometry assay as<br/>described below.

1. Measure cell density of induced NbLib yeast (1 OD600 ≈ 1.5 x 107 cells/mL)<br/>2. Add 500 μl of ‘selection buffer’ to two microcentrifuge tubes<br/>3. Pipette approximately 1×106 induced yeast into each microcentrifuge tube and<br/>centrifuge for 1 min at 3500 × g, 4 °C<br/>4. Aspirate supernatant and resuspend pellet in 100 μl selection buffer<br/>5. To one tube add ~0.5 μg of anti-HA antibody labeled with AlexaFluor647 or<br/>AlexaFluor488 (to check for the percentage of cells expressing nanobody) and 1<br/>μM final concentration of your protein labeled with a spectroscopically distinct<br/>dye (to check if your antigen binds nonspecifically to yeast). Leave the other<br/>tube unstained as a control<br/>6. Rock both tubes at 4 °C for 15 minutes<br/>7. Spin down cells for 1 minute at 3500 × g, 4 °C<br/>8. Aspirate supernatant and resuspend cells in 500 μL of selection buffer<br/>9. Spin down cells as before and aspirate supernatant<br/>10. Resuspend cells in 100 μl of selection buffer and assess nanobody expression<br/>level on flow cytometer using unstained sample to set gates

Note: The maximum number of expressing cells from the naïve library is ~25%. The<br/>typical number of expressing cells is ~15-20%. If expression is on the low end (~8-12%)<br/>consider increasing the number of cells used for nanobody selection to compensate.<br/>Example flow cytometer yeast gating:

Nanobody Selection<br/>The selection process below is at the heart of identifying clones that bind to your target<br/>protein. This protocol describes how to separate antigen binding nanobody clones from<br/>non-binders using magnetic cell sorting in two major steps: a “pre-clear” or negative<br/>selection step, followed by a positive selection step. These are usually done in series on<br/>the same day. The pre-clear step serves to deplete the library of any nanobodies that<br/>bind to things like secondary antibodies, magnetic beads, etc. The positive selection<br/>step isolates clones that bind to your antigen. After selection, you can expand your<br/>enriched cells and repeat the process until binders are highly enriched.<br/>11. Spin down a suitable number of cells to have at least 10-fold over library<br/>diversity. For the naïve library (NbLib) you will need 5 × 109 cells. After one or<br/>more rounds of selection a smaller number of cells may be used based on<br/>estimated diversity. We recommend always using at least 1 × 107 cells, since<br/>smaller numbers are difficult to manipulate. Perform centrifugation for 3<br/>minutes at 3500 × g, 4 °C<br/>12. Aspirate media and resuspend yeast via pipetting in 10 mL of chilled selection<br/>buffer<br/>13. Spin down yeast for 3 minutes at 3500 × g, 4 °C and aspirate supernatant<br/>14. Resuspend yeast in 4.5 mL of selection buffer<br/>15. Begin preclear to remove off-target binders from the naïve library by adding 500<br/>μl of the ‘Miltenyi anti-fluorophore beads’ to the resuspended yeast (If using a<br/>secondary for selection, add that to the preclearing reaction as well. ~200 nM is<br/>typically sufficient to remove secondary binders)<br/>16. Rock/Rotate slowly at 4 °C for 40 minutes<br/>17. Approximately 20 minutes prior to the conclusion of incubation, place an ‘LD<br/>column’ on the Miltenyi MACS magnet. Put a 15 mL sterile falcon under the<br/>column and place a 50mL falcon cap over the top of the column to maintain<br/>sterility. Add 5 mL of selection buffer to the LD column to allow it to equilibrate<br/>before the next step. It will take ~20 minutes to flow through completely<br/>18. Spin down yeast for 3 minutes at 3500 × g at 4 °C and aspirate buffer and<br/>unbound beads<br/>19. Resuspend by pipetting in 5 mL of selection buffer.<br/>20. Flow yeast over equilibrated LD column, collect flowthrough in new sterile 15mL<br/>centrifuge tube<br/>21. Flush out remaining cells by washing column with an additional 2 mL of<br/>selection buffer<br/>22. Remove LD column from magnet and discard<br/>23. Spin down flow through at 3500 × g at 4 °C and aspirate supernatant<br/>24. To resuspended yeast, add 5mL selection buffer containing fluorophore labeled<br/>antigen at a 1-1.5 μM concentration (if using a secondary to stain – preincubate<br/>the secondary with antigen before staining yeast and use a molar concentration<br/>of 2:3 secondary to antigen)<br/>25. Rock or rotate slowly at 4 °C for 1 hour<br/>26. Spin down yeast for 3 minutes at 3500 × g at 4 °C and aspirate supernatant<br/>27. Resuspend yeast in 4.5 mL of selection buffer and 500 μl of anti-fluorophore<br/>beads<br/>28. Rock or rotate slowly at 4 °C for 20 minutes<br/>29. Approximately 5 minutes before end of incubation, place an ‘LS column’ on the<br/>Miltenyi MACS magnet. Put a 15 mL sterile falcon tube under the column and<br/>place a 50mL cap over the top of the column to maintain sterility. Keep plunger<br/>wrapped in package for later use. Add 5 mL of selection buffer to the LS column<br/>and allow it to equilibrate before the next step. It will take ~5 minutes to flow<br/>through completely<br/>30. Spin down yeast for 3 minutes at 3500 × g at 4 °C and aspirate buffer and<br/>unbound beads<br/>31. Resuspend yeast by pipetting in 3 mL of selection buffer and spin down as above<br/>32. Aspirate supernatant and resuspend yeast in 3 mL of selection buffer<br/>33. Remove 50 μl “pre-LS” aliquot of resuspended yeast and set aside for later<br/>analysis. Keep aliquot on ice.<br/>34. Flow yeast over the equilibrated LS column and collect flow through in a new<br/>sterile 15 mL centrifuge tube<br/>35. Wash column with 8 mL of selection buffer&nbsp;<br/>36. Record approx. volume of flow through and remove 50 μl “flow through” aliquot<br/>for later analysis.<br/>37. Remove LS column from magnet and place over fresh sterile 15 mL centrifuge<br/>tube<br/>38. Add 5 mL of selection buffer to column and immediately use plunger to elute<br/>cells<br/>39. Remove 50 μl “elution” aliquot from eluted cells and set aside for later analysis.<br/>Keep aliquot on ice.<br/>40. Spin down elution at 3500 × g for 3 min at 4 °C and aspirate buffer taking care<br/>not to disturb the pellet<br/>41. Resuspend yeast in 3 mL of –TRP +glucose in a 14 mL Falcon culture tube<br/>42. Shake at 30 °C for about 24 hours to recover.<br/>43. Use flow cytometry to compare the number of yeast from pre-LS and elution<br/>aliquots and use this ratio to get an estimate of the reduction in diversity of the<br/>library.<br/>Next steps: After recovering yeast, you can reinduce in galactose medium for<br/>another round of selection, and iterate until you isolate binders. It’s a good idea to<br/>use a different fluorophore for each selection round. We typically perform two<br/>MACS selections followed by a FACS sort to isolate binders.<br/>For a FACS sort, you can prepare your yeast by staining with antigen exactly as<br/>described above. The details of how to sort will depend on the specific project and<br/>sorting equipment being used. Once your selected library has a high proportion of<br/>active clones (usually 20% or more of the total cells), you can move on to screening.<br/>For screening, plate sorted yeast on –TRP plates for single clones. Grow single<br/>clones in a sterile deep-well 96-well plate, with one clone per well. Then induce<br/>expression and stain clones to identify those with the highest activity. Finally, a<br/>yeast miniprep or colony PCR can be used to isolate nanobody DNA for analysis and<br/>recombinant expression. Note that not every clone with activity on yeast retains<br/>activity in recombinant form. A large majority of clones will show activity in both<br/>formats, but there are some exceptions.<br/>Appendix<br/>3.8 g of –Trp drop-out media supplement (US Biological)<br/>Yglc4.5 –Trp (1 liter)<br/>6.7 g Yeast Nitrogen Base<br/>10.4 g Sodium Citrate<br/>7.4 g Citric Acid Monohydrate<br/>10 mL Pen-Strep (10,000 units/mL stock)<br/>20 g glucose<br/>Adjust pH to 4.5; Sterifilter (or sterifilter a stock of 20% glucose and add with antibiotic<br/>after autoclaving).<br/>3.8 g –Trp drop-out media supplement (US Biological)<br/>–Trp (+glucose or +galactose - 1 liter)<br/>6.7 g Yeast Nitrogen Base<br/>10 mL Pen-Strep (10,000 units/mL stock)<br/>20 g glucose or galactose (glucose for normal growth and galactose for induction of<br/>nanobodies)<br/>Adjust pH to 6; Sterifilter (or sterifilter a stock of 20% glucose or galactose and add with<br/>antibiotic after autoclaving).<br/>20 g Bacto Peptone<br/>YPAD (1 liter)<br/>20 g Glucose<br/>10 g Yeast Extract<br/>18 mg Adenine<br/>10 mL Pen-Strep (10,000 units/mL stock)<br/>20 mM HEPES pH 7.5<br/>Selection buffer*<br/>150 mM sodium chloride<br/>0.1% (w/v) bovine serum albumin<br/>5 mM maltose<br/>Sterile filter before use.<br/>*Many other buffers will work equally well, depending on your protein you may need to<br/>modify this. For GPCRs for instance, we usually add 0.1% (w/v) lauryl maltose neopentyl<br/>glycol detergent.<br/>No pH adjustment needed. Sterifilter or autoclave as appropriate<br/>MACS accessories catalog numbers<br/>Anti-Cy5/Anti-Alexa Fluor 647 MicroBeads Miltenyi Biotec Cat# 130-091-395<br/>Anti-FITC MicroBeads Miltenyi Biotec Cat# 130-048-701<br/>LD Columns Miltenyi Biotec Cat# 130-042-901<br/>LS Columns Miltenyi Biotec Cat# 130-042-401

'''pYDS649HM'''

Library vector sequence: Nb.BV025 in pYDS649HM 8584 bp. Selectable in E. coli with ampicillin and in yeast by Trp dropout. Sequence with Gal1 promoter primer (“AATATACCTCTATACTTTAACGTC”).



GGTACCCGACAGGTTATCAGCAACAACACAGTCATATCCATTCTCAATTAGCTCTACCA CAGTGTGTGAACCAATGTATCCAGCACCACCTGTAACCAAAACAATTTTAGAAGTACTT TCACTTTGTAACTGAGCTGTCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTG GATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACCATATACATATCC ATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTA TCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGC TATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGAC TCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGC GCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAG GAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATGAACGAATCAAATTAACAA CCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGA AGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAACTGCATAACCACTTTA ACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATC AACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGAT CGAATTCAACCCTCACTAAAGGGCGGCCGCC

Mating factor alpha leader

ATGAGATTCCCATCTATCTTCACCGCTG TTTTGTTCGCTGCTTCTTCTGCTTTGGCTGCTCCAGCTAACACCACCACCGAAGACGAA ACCGCTCAAATCCCAGCTGAAGCTGTTATCGACTACTCTGACTTGGAAGGTGACTTCGA CGCTGCTGCTTTGCCATTGTCTAACTCTACCAACAACGGTTTGTCTTCTACCAACACCAC CATCGCTTCTATCGCTGCTAAGGAAGAAGGTGTTCAATTGGACAAGAGAGAA

MRFPSIFTAVLFAASSALAAPANTTTEDETAQIPAEAVIDYSDLEGDFDAAALPLSNSTNNGLSSTNTTIASIAAKEEGVQLDKRE

GCTAGC GCA

ASA

Nanobody coding sequence

CAGGTGCAGCTGCAGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGGCAGCCTG CGCCTGAGCTGCGCGGCGAGCGGCTCTATTTCTCTGTACGCTGAAATGGGCTGGTATC GCCAGGCGCCGGGCAAAGAACGCGAACTTGTTGCCGGTATTACTCGTGGTACTACTAC CTATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAAC ACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCG CGGCTGACTACGTTCAGGGTAAATCTCGTGTTATCCCGCATTACTATTGGGGCCAGGG CACCCAGGTGACCGTGAGCAGC

QVQLQESGGGLVQAGGSLRLSCAASGSISLYAEMGWYRQAPGKERELVAGITRGTTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADYVQGKSRVIPHYYWGQGTQVTVSS

GGATCC

HA epitope tag

TACCCATACGATGTTCCAGATTACGCT

649 stalk sequence

CAA ACATCCTCACCCTCTACAGAAGCATCAGTATCAACCTCTAGTACCTCTTCCTCTGCTTCA CAATCATCTGACCCAACTACAACATCTTCGTCCAGTTCATCGTCTTCCCCATCTTCCCAA TCTGAGGAAATTTCATCATCACCGACGGTCTCAACTACACCATCAACTTCTTCATCATCT TCCTCAATGACTTCAACCACCACAACAAAGTCAATCTCAACTTCCACTACAAGTTCAGC TCCAGTTACAGATGTGACAGTTTCCTCATCGCCTAGTAAATCTACCTCTACTTCGACAA GTACAGAAACATCTAAAACACCTACTTCAATGACAGAGTATACATCTAGTACATCGATA ATTTCGACTCCAGTTAGTCACTCGCAGACAGGTTTGTCGGCTTCATCAAGTTCATCATC TACAACATCCGGTTCTTCGTCCACTAAATCAGAAAGTTCGACAACATCTGGCTCTTCCC AGTCCGTGGAATCAACCTCCAGCCACGCCACTGTTCTTGCTAATTCCGCAGAAATGGTC ACAACATCCTCTAGTTCATCCTCAACATCCGAAATGTCATTAACTAGTACTGCTACCAG TGTACCAGTCTCATCTAGTAGCAGTACGACATATTCTACTAGCGCATCTACACAAGCCG TCACTACAACATCTTCTTCCACTGTATCTACAACTTCTTCTAGTACAACGTTAACAAGCG CATTCACACATTCTTCAACCACATCGTCCGACCAGCCACCCAGCGACACTACAAGTCCA TCTACGACACACGAACCTCATGTAACCACTCAGACGTCATCAGAAACATCTTCTTCTAA GTCATCTTCTACTTCTTCTTCAAGTACATCTCAAACCTCTGAGTCTGCAACACCATCCGA TTCCGTATCACCTGGAAGTTCTACATCAACATCTTCTAGTAGCACTTCTACTTCCACTTC TATTTCCAGTGGAGAAACGACAACTTCTTCTTCTTCATCATCTGCCACGACCACTTCTAA CAGCGCAACCTTGTCAGTCTCTACCACACAAACTTCGATTGAAGCCAGTTCATCTACTA CATCTACATCTAGTTCAACAATTACAACTTCAAGTAGTAGCGCTCACATATCGTCGAAA TCTCAATCTAGTATTACCTATCCCTCTTCCTCGACATCTTCATCTACATCGTCCTCAATTT CTAGCGAATCTGAAAGTTTTGAATCGACATCAGCAGAAGATGCTCCATCAACAGCACC TTCATCAAGTGTCTCTTCTAAGAGTTCTACCTCTACAACATCAAGCACATCGACATCTTC AAGCACTCCATCTCCATCACCATCTTCCGTGAGTTCTTCCTCCACCAGCTCATTGACAAC TTCTGCTGTATCAACACCAGCTACCTCTCATTCTCAAAGTACTGTAGTAACCACCACTA CTATTACTACATCAACAGGTCCAGTGATGTCTACGACAACAGCTTATTCTTCTAGTTCT ACTAGCAGCTCGGAATCTTCTGAGGTTCAGTCTGTCATGTCATCTACGCCTAGTTCAAC ATCAACAACAACCAGTTCGGAATCTACTTCATCTAGCTCCACAGCTTCTACCTCACCAT CAACCTCGCAAACTTTCGAAACTTCTCCTACTATAGGAGGTGTCCCCTCAACCACTTCA TTTGTCTCTACGCCAACAACGAAATTGTCGCACACTACTTCCACTATGACAGCACAGTC CGATAGTAAGTCTACCCACTCCTCAAGCACATCGACAGAAGATAAATCATCCACTGCTT CTGCAGTTGACGAAAGCACTACAACATCCACTTCCACGGAGTCTACTACATCAGTAACA TCAGGCACCTCCCATTCCGCTAAAGAATCTTCGTCAAATTCTAAGGTGTATAGTTCACA GACAGCACACTCATCCATAAGTGTTGCATCATCACCTAGTACAAAG

GGCGCC

CAAATC CAATCTTCTATGGTTGAAATCTCTACCTACGCTGGTTCTGCTAACTCTGTTAACGCTGG TGCTGGTGCTGGTGCTTTGTTCTTGTTGTTGTCTTTGGCTATCATC



TAATGATTAATTAA CTCGAGATCTGATAACAACAGTGTAGATGTAACAAAATCGACTTTGTTCCCACTGTACT TTTAGCTCGTACAAAATACAATATACTTTTCATTTCTCCGTAAACAACATGTTTTCCCAT GTAATATCCTTTTCTATTTTTCGTTCCGTTACCAACTTTACACATACTTTATATAGCTATT CACTTCTATACACTAAAAAACTAAGACAATTTTAATTTTGCTGCCTGCCATATTTCAATT TGTTATAAATTCCTATAATTTATCCTATTAGTAGCTAAAAAAAGATGAATGTGAATCGA ATCCTAAGAGAATTGAGCTCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGC CGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTG CAGCACATCCCCCCTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCC TTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCGACGCGCCCTGTAGCGGCGCA TTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCC TAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCC GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTC GACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGA CGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAA ACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCC GATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTA ACAAAATATTAACGTTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCG GTATTTCACACCGCAGGCAAGTGCACAAACAATACTTAAATAAATACTACTCAGTAATA ACCTATTTCTTAGCATTTTTGACGAAATTTGCTATTTTGTTAGAGTCTTTTACACCATTT GTCTCCACACCTCCGCTTACATCAACACCAATAACGCCATTTAATCTAAGCGCATCACC AACATTTTCTGGCGTCAGTCCACCAGCTAACATAAAATGTAAGCTTTCGGGGCTCTCTT GCCTTCCAACCCAGTCAGAAATCGAGTTCCAATCCAAAAGTTCACCTGTCCCACCTGCT TCTGAATCAAACAAGGGAATAAACGAATGAGGTTTCTGTGAAGCTGCACTGAGTAGTA TGTTGCAGTCTTTTGGAAATACGAGTCTTTTAATAACTGGCAAACCGAGGAACTCTTGG TATTCTTGCCACGACTCATCTCCATGCAGTTGGACGATATCAATGCCGTAATCATTGAC CAGAGCCAAAACATCCTCCTTAGGTTGATTACGAAACACGCCAACCAAGTATTTCGGA GTGCCTGAACTATTTTTATATGCTTTTACAAGACTTGAAATTTTCCTTGCAATAACCGG GTCAATTGTTCTCTTTCTATTGGGCACACATATAATACCCAGCAAGTCAGCATCGGAAT CTAGAGCACATTCTGCGGCCTCTGTGCTCTGCAAGCCGCAAACTTTCACCAATGGACCA GAACTACCTGTGAAATTAATAACAGACATACTCCAAGCTGCCTTTGTGTGCTTAATCAC GTATACTCACGTGCTCAATAGTCACCAATGCCCTCCCTCTTGGCCCTCTCCTTTTCTTTT TTCGACCGAATTAATTCTTAATCGGCAAAAAAAGAAAAGCTCCGGATCAAGATTGTAC GTAAGGTGACAAGCTATTTTTCAATAAAGAATATCTTCCACTACTGCCATCTGGCGTCA TAACTGCAAAGTACACATATATTACGATGCTGTCTATTAAATGCTTCCTATATTATATA TATAGTAATGTCGTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTA AGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCC CGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTT TTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTA TAGGTTAATGTCATGATAATAATGGTTTCTTAGGACGGATCGCTTGCCTGTAACTTACA CGCGCCTCGTATCTTTTAATGATGGAATAATTTGGGAATTTACTCTGTGTTTATTTATTT TTATGTTTTGTATTTGGATTTTAGAAAGTAAATAAAGAAGGTAGAAGAGTTACGGAAT GAAGAAAAAAAAATAAACAAAGGTTTAAAAAATTTCAACAAAAAGCGTACTTTACATA TATATTTATTAGACAAGAAAAGCAGATTAAATAGATATACATTCGATTAACGATAAGT AAAATGTAAAATCACAGGATTTTCGTGTGTGGTCTTCTACACAGACAAGATGAAACAA TTCGGCATTAATACCTGAGAGCAGGAAGAGCAAGATAAAAGGTAGTATTTGTTGGCGA TCCCCCTAGAGTCTTTTACATCTTCGGAAAACAAAAACTATTTTTTCTTTAATTTCTTTT TTTACTTTCTATTTTTAATTTATATATTTATATTAAAAAATTTAAATTATAATTATTTTTA TAGCACGTGATGAAAAGGACCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCC CTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCT GATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGT CGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCT GGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACT GGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCA AGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCA GTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCA TAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA GGAGCTAACCGCTTTTTTTCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGG AACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGC AATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGC AACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGC CCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGC GGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACA CGACGGGCAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTG CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATT GATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCT CATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAA AGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACA AAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTT TCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAG CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCT AATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGAC TCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGC ACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC ATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCG GCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGGAACGCCTGGTATC TTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCG TCAGGGGGGCCGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG CCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA ACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCG CAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCC CGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCG GGCAGTGAGCGCAACGCAATTAATGTGAGTTACCTCACTCATTAGGCACCCCAGGCTT TACACTTTATGCTTCCGGCTCCTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCAC ACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGGAATTAACCCTCACTAAAGGG AACAAAAGCTG
[[Category:Protocols]]