Team:Harvard/Template:NotebookData2

June 14

• Made four LB-based media solutions, and later created glycerol stocks from these and placed in -80⁰C freezer
  • Selection strain (ΔHis3ΔPyrFΔrpoZ) in 3 mL LB and 3µL of 1000x Tet solution stock
  • Selection strain (ΔHis3ΔPyrFΔrpoZ) in 3 mL of LB only solution stock (control)
  • Kan cassette (pZE22G) in 3 mL of LB solution stock and 3 µL of kanamycin solution stock
  • Lambda Red (pKD42) in 3 mL of LB and 3 µL Amp solution stock
    • For all of these stocks, we tried to grow all to mid-log and then place them in 1,200 µL of culture and 300 µL of 80 % glycerol solution (This is the correct protocol for creating a glycerol stock; refer to June 9th)
    • We were only able to get the kan cassette to mid-log and created glycerol stock of the kan cassette
    • Observations included contamination of a pKD46 liquid culture, and we are leaving Lambda Red and both solutions with the selection strain for overnight growth
• Ran 1% gel (150V) with the rest of the OZ123 and OZ052 overhang PCR samples
  • Used better ladder today, less the 1 kb ladder
  • Bands followed the same pattern as the gel run on 6/13/11
• Used gel extraction to obtain the correct OZ123 and OZ052 PCR product from the gel
  • Used Qiagen quick gel extraction kit
  • OZ052 (from undiluted lane): 7.0 ng/µL, 260/280=2.42 (Note: this sample had a strange yellow substance in the column, may have been contaminated)
  • OZ052 (from undiluted lane): 12.0 ng/µL, 260/280=2.02
  • OZ052 (from 1:10 dilution): 10.8 ng/µL, 260/280=2.04
  • OZ052 (from 1:10 dilution): 15.2 ng/µL, 260/280=1.82
  • OZ052 (from 1:100 dilution): 20.6 ng/µL, 260/280=2.17
  • OZ123 (from undiluted lane): 6.3 ng/µL, 260/280=2.17
• PCR the backbone fragment of the plasmid using Wolfe_R and Wolfe_L primers and a lower annealing temperature than before due to the lower melting point of Wolfe_L
  • Reagents:
    • 22µL Invitrogen Platinum PCR supermix
    • 1µL template from a 1 in 100 dilution (1 ng)
    • 1µL Wolfe_F and 1µL Wolfe_R
  • 94°C for 30 s
  • 94°C for 30 s
  • 53°C for 30 s
  • 70°C for 5 min
    • Previous three steps repeat 30 times
  • 70°C for 5 min
  • 4°C forever
• Performed a restriction enzyme digestion on the selection construct plasmid using EcoRI to test for presence/absence of inserted selection construct
  • 1µL EcoR1
  • 1µL buffer 4
  • 2µL backbone plasmid
  • 6µL ddH2O
  • Incubate at 37 degrees for 90 min
  • There is only one EcoR1 site (GAATTC) in the plasmid, so we should see 1 band at about 5kb
• Ran a gel (1%, 170 V) on the backbone fragment (Wolfe primers) PCR product and restriction digestion result
  • Observations: the EcoR1 digest produced the expected band of around 5kb. The backbone did produce a 5kb band but also had a secondary smaller product, perhaps due to one of the primers annealing to a sequence that is a close match to its target.
• Began gradient PCR on the selection strain backbone with Wolfe_R and Wolfe_F primers because of the large difference in melting temperatures between the two
  • Set the annealing temperatures within the PCR to go from 50-57 C and ran with 5 minute extension phases at 70 C
  • Ran overnight

Today's Gel Images

Ultramer Overhang 6/14/11

Digestion and PCR Backbone on Selection Construct Plasmid 6/14/11

June 14 - Bioinformatics

We finished writing the generate function, and now have a working sequence generator. We also began more in-depth research into the 2011 work by Persikov which deals with how the zinc finger binds to DNA. He predicts several relations which we should be able to test.

Persikov sent us his SVM code (used to calculate the probability of a sequence binding to given DNA), so we also worked on adapting this to use when narrowing our sequences to those most likely to work.

• There are four canonical amino acid-base interactions involved in zinc finger interactions.
  • Amino acids in positions -1, 3, and 6 on the helix are known to interact with the 3 bases of the triplet. In addition, the amino acid in position 2 interacts with the upstream base of the complementary strand (Klug 2010).
  • In addition, Persikov (2011) has proposed novel interactions between these four amino acids based on his analysis of zinc finger binding data upto 2005.
  • Persikov uses the information between these four amino acid-base interactions in his SVM, to determine whether a finger would be a good binder to a particular DNA sequence.
• In order to use his program, we need to convert the helices that are created by the generator into a format the SVM accepts.
  • The SVM only considers the four canonical interactions. It assigns a numerical value to each possible amino acid-base combination. The program that converts our data into a format the SVM accepts creates a string with these numerical values based on Persikov's key. (See this page for more details on this program.)

June 15th

Gradient PCR: 5µL of each PCR product were run on a 1% gel. No bands appeared: the PCR appears to not have worked.

Selection construct: bacteria containing the selection construct (plasmid containing ZF, omega subunit, ZFB, His3, URA3, etc.) was made into a glycerol stock (see 6/14 and 6/9), miniprepped, and used for PCR:

• Miniprep: used Qiagen kit
  • 82.5ng/µL, 260/280=1.99
  • 91.1ng/µL, 260/280=2.01
  • 77.9ng/µL, 260/280=1.98
• PCR:
  • PCR used to amplify section of plasmid containing zinc finger binding site, weak promoter, His3, and URA3 (with homology to join it to kan cassette)
  • Reagents
  • zinc finger binding site and weak promoter, selection construct plasmid:
    • 1µL ZFB-wp-f (5µM) (made by 1:20 dilution of 100µM stock)
    • 1µL ZFB-wp-hisura-r (5µM) (made by 1:20 dilution of 100µM stock)
    • 1µL selection construct (1:100 dilution of overnight culture)
    • 22.5µL of invitrogen's Platinum PCR SuperMix
  • PCR used to amplify the kan cassette
  • Reagents
  • KAN cassette, pZE22g plasmid:
    • 1µL hisura-kan-f (5µM) (made by 1:20 dilution of 100µM stock)
    • 1µL kan-r (5µM) (made by 1:20 dilution of 100µM stock)
    • 1µL pZE22g (1:100 dilution of glycerol stock culture)
    • 22.5µL of invitrogen's Platinum PCR SuperMix
  • Parameters:
• 1) 94°C for 2 min (denature template, activate enzyme)
• 2) 94°C for 30 sec (denature)
• 3) 53°C for 30 sec (anneal)
• 4) 72°C for 2 min (extend)
• 5) Repeat 2-4 for 25 cycles total
• 6) 72⁰C for 5 min
• 7) 4°C forever

PCR Purification:

• Used the Qiagen PCR purification kit and instructions in order to purify the Kan cassette and selections construct PCR products
  • Nanodrop the purification results and observed 3 ng/µL for Kan cassette and 29.8 ng/µL for ZFB-wp-His3: purification did not work well, especially for the kan

Selection strain (ΔHis3ΔPyrFΔrpoZ):

• saturated overnight culture was inoculated again: 3mL LB, 3µL tet, 30µL of overnight culture, at 37C until mid-log
• glycerol stock
• For transformation tomorrow we grew up pKD42 in 3 mL of LB, 1.5 µL of ampicillin(2000x) and one colony at 30 C
  • Also grew up more of the selection strain so it will be ready for electroporation transformation

Gel

• Ran gel with Kan cassette and selection construct (Binding site, His3, and URA3)
  • Observations successful and image below
  • Used 1 kb plus ladder

Kan and ZFB-wp-his constructs 6/15/11

PCR Overlap

• since the purification was not very successful, we used 3µL saved from the original PCR product
• Procedure
  • 25µL of 2x Phusion Master Mix
  • 1 µL of ZFB-wp-HisURA-R (100µM)
  • 1µL of HisURA-Kan-F (100µM)
  • 21 µL of water
  • 1 µL Kan template and 1 µL of ZFB-wp-His3-URA3
    • 4 tubes
      • Both undiluted
      • Both 1:10 dilution
      • Both 1:100 dilution
      • Both 1:1000 dilution
• Protocol
  • 98 C for 30 s
  • 98 C for 10 s
  • 53 C for 30 s
  • 72 C for 3 min
  • Repeat steps 2-4 for 24 more cycles
  • 72 C for 5 min
  • 4 C 4EVA!!!

June 15th - Bioinformatics

• We continued research into Persikov's and others' work on binding.

• We worked on using the OPEN data to test Persikov's binding-predicting program: SVM

• Justin continued work on a sequence-finding program, the most up to date version can be found in the Dropbox under code/zfsitefinder.html.
• Justin and Will found 10 candidate sequences across 4 diseases that hopefully should encompass a good amount of diversity in terms of expanding the ZF library. These sequences can be found in the table below, with more details here. The most up to date version can be found in the Dropbox under sequences/Target Loci Sequences.

• We collected 15 alternative zinc finger backbones (different from zif268 backbone) and their corresponding base sequences. Many of these were from Persikov 2011 and all binding sequences were confirmed on the Protein Data Bank website at http://www.pdb.org/pdb/home/home.do. The zinc finger PDB ID's and related links are:

June 16th

• So there was totally a crazy bee hive outside today!!

Glycerol Stock pKD42

• Grew up pKD42 in 30 C and once reached mid-log created glycerol stock and placed in -80 refrigerator

Overlap PCR gel

• Ran gel to test if overlap PCR that ran through the night worked, and it did not
  • Used PCR product without purification which gives good explanation for why it didn't work

• PCR: Since the PCR done the previous day (6/15) we made a back up PCR using phusion mastermix (Finnzyme)
  • PCR used to amplify section of plasmid containing zinc finger binding site, weak promoter, His3, and URA3 (with homology to join it to kan cassette)
  • Reagents
  • zinc finger binding site and weak promoter, selection construct plasmid:
    • 1µL ZFB-wp-f (100µM) (taken directly from the primer tube)
    • 1µL ZFB-wp-hisura-r (100µM) (taken directly from the primer tube)
    • 2µL selection construct (1:100 dilution of overnight culture)
    • 25µL Phusion High-Fidelity PCR Master Mix
    • 21µL distilled water (for total volume of 50µL)
  • PCR used to amplify the kan cassette
  • Reagents
  • KAN cassette, pZE22g plasmid:
    • 1µL hisura-kan-f (100µM) (taken directly from the primer tube)
    • 1µL kan-r (100µM) (taken directly from the primer tube)
    • 2µL pZE22g (1:100 dilution of glycerol stock culture)
    • 25µL of Phusion High-Fidelity PCR Master Mix
    • 21µL distilled water (for total volume of 50µL)
  • Parameters:
    • 1) 94°C for 2 min (denature template, activate enzyme)
    • 2) 94°C for 30 sec (denature)
    • 3) 53°C for 30 sec (anneal)
    • 4) 72°C for 2 min (extend)
    • 5) Repeat 2-4 for 25 cycles total
    • 6) 72⁰C for 5 min
    • 7) 4°C forever
• Ran PCR product on a gel: bands of the correct size were observed, though the kan band was much fainter than the ZFB-wp-his3

• Repeat PCR (to get a higher concentration of the Kan cassette and ZFB-wp-his3 constructs)
  • same as the above backup PCR (since it was successful), but to a 4x total volume of 200µL, compared to 50µL
  • PCR products were run on a gel: the correct bands were observed--see image below ("second gel")
  • PCR product purification: followed Qiagen kit instructions. Strangely, the conc. was 64.6 ng/µL for Kan, purity 2.09 (260/280) and for ZFB-wp-his3, the conc. was 23.7ng/µL and the purity was 1.92 (260/280).

Kan cassette and ZFB-wp-his3 constructs second gel 6/16/11

Overlap PCR: used the kan cassette (64.6 ng/µL) and ZFB-wp-his3-ura3 (23.7 ng/µL) purified above

• 1 µL of kan and 1 µL of ZFB-wp-his3-ura in each tube according to the following conditions:
  • two tubes: undiluted
  • two tubes: both diluted 1 in 10
  • two tubes: both diluted 1 in 100
• 12.5µL Phusion master mix
• 8 µL ddH2O
• primers: 1.25 µL ZFB-wp-hisura_r (10 µM) and 1.25 µL hisura-kan_f (10 µM)
  • we tried two different reaction types: one added the primers as usual before starting the PCR reaction, the other added the primers after 10 PCR cycles (allowing the polymerase to first use the overlapping kan and ZFB to elongate, and then the primers)
• Parameters for PCR starting with primers:
  • (PCR machine 5, program name EXT3KB in IGEM folder)
  • 1) 98°C for 1 min (denature template, activate enzyme)
  • 2) 98°C for 15 sec (denature)
  • 3) 65°C for 15 sec (anneal)
  • 4) 72°C for 2 min (extend)
  • 5) Repeat 2-4 for 30 cycles total
  • 6) 72⁰C for 5 min
  • 7) 4°C forever
• Parameters for PCR starting without primers:
  • 1) 98C for 1 min
  • 2) 98C for 15 sec
  • 3) 65C for 15 sec
  • 4) 72C for 1 min
  • 5) back to step 2 for 10 cycles (PCR paused after 10 and primers added)
  • 6) 98C for 15 sec
  • 7) 65C for 15 sec
  • 8) 72C for 2 min
  • 9) back to step 6 for 20 cycles
  • 10) 72C for 5 min
  • 11) 4C forever

Transformation

• Used the selection strain (ΔHis3ΔPyrFΔrpoZ) cells at mid-log and attempted to place lambda red (pKD42) plasmid into the cell
• Procedure
  • Keep on ice through out whole procedure before use of the electroporation machine
  • Spin 1.5 mL of mid-log cells at 4 C for 1 minute at 18000 rcf (we created two tubes through the following steps)
  • Discard supernatant and resuspend with 1 mL of cold water
  • Spin again and repeat for a second water wash
    • With each wash, try to get as much supernatant out as possible(even use pipette) because don't want salts to interfere with the electrical pulse
  • Resuspend pellet with 50 µL of cold water
  • Add 1 ng of pKD42 to one of the tubes and 45 ng of pKD 42 to the other
  • Take all of the liquid in each tube and place in two separate cuvettes for electroporation
    • Make sure the electroporation machine is on the right setting (for the cuvettes we used today it was "Ec2")
    • Wipe off all water on the side of the cuvette
  • Have 1 mL of LB in hand and after pulsing, immediately put LB in cuvette
  • Transfer to culture tube and place in 30 C for 2 hours
  • Make 4 LB/amp plates and spread E. coli using glass beads:
    • Plate 1: 10 µL of 1 ng culture
    • Plate 2: take 700 µL of 1 ng culture, spin down and remove supernatant, resuspend in about 30 µL of LB and plate
    • Plate 3: 10 µL of 45 ng culture
    • Plate 4: take 700 µL of 45 ng culture, spin down and remove supernatant, resuspend in about 30 µL of LB and plate
  • Grow overnight at 30 C

PCR to confirm knockouts of selection strain

• this PCR was to confirm that the ΔHis3ΔPyrFΔrpoZ was indeed a knockout for the His3, PyrF, and rpoZ genes
• each primer set was used for two conditions: wild-type (we used a pKD42 culture) and knockout (ΔHis3ΔPyrFΔrpoZ culture, left over from transformation)
• 1 µL of either wt or ko template, diluted 1:20
• 12.5 µL Phusion master mix
• 1.25 µL of each 10µM primer:
  • test for His3:
    • 1)His3_F, His3_R
    • 2)His3_F, His3_internalR
  • test for PyrF:
    • 3)PyrF_F, PyrF_R
    • 4)PyrF_F, PyrF_internalR
  • test for rpoZ:
    • 5)rpoZ_F, rpoZ_R
    • 6)rpoZ_F, rpoZ_internalR
  • test for Zeocin (there are two primer sets because we don't know what orientation the Zeocin gene is in)
    • 7)Zeocin_R, rpoZ_F
    • 8)Zeocin_R, rpoZ_R
• ddH2O up to 25 µL
• Parameters:
  • 98 C for 5 min
  • 98 C for 10 sec
  • 65 C for 25 sec
  • 72 C for 45 sec
  • cycle 30 times
  • 72 C for 5 min
  • 4 C forever
• Results: ran PCR products out on 1% gel (see below). There were some nonspecific bands, but the PyrF and rpoZ genes do appear to be knocked out in the selection strain. His3, however, looks like it's still present--we'll test again to confirm.

June 16 - Bioinformatics

• Research Targets

1. Clinically relevant targets
2. Existing ZFs that bind under-represented triplets

Updating our programs

• Many of our current programs currently look at overall data or data based on specific DNA triplets (for example: 'GAT' or 'AAA'). However, in order to more easily understand some of the patterns that occur in the datasets, we want to examine broader subsets of data. For example, do different patterns appear when looking at fingers that bind to 'GNN' triplets versus 'NGN' triplets (where 'N' represents any of the 4 bases)?
  • We added the capability for our programs to accept inputs with the variable 'N' by using regular expressions.
    • We can now create lists of the zinc fingers that bind to any triplet, and create interaction matrices and frequency tables for any triplet input.

June 17th

Update on selection strain knockout status: We are trying to reach Addgene to check how His3 was knocked out---instead of deleting the gene, they may have simply introduced an early stop codon. If that's the case, our gel would have the correct bands because the primers we designed can only show whether a deletion or insertion was in that locus.

Transformation results: successful!!

• The only plate with colonies was the one plated with 700 µL of cells transformed with 45 ng of pKD42
• Chose a colony to grow in 3mL LB, 1.5µL amp, 30C; make glycerol stock with mid-log cells
• Plate with colonies at 4C

Miniprep of pZE22G: (to have the plasmid containing the kan cassette on hand)

• used 2 tubes of 1.5mL overnight culture, followed Qiagen kit instructions
• 38.0 ng/µL, 260/280=1.99
• 27.8ng/µL, 260/280=2.02

Overlap PCR gel and extraction: 1%, 150V

• Results: adding the primers in after 10 cycles was much more successful than adding them at the beginning, and all three dilutions showed the expected product band (about 2.5kb). The rest of the 1:10 dilution will be run on a gel and extracted.
• 11.2ng/µL, 260/280=2.10

June 17 - Bioinformatics

Goals

1. Make BB Database in program-readable format ✓
2. Edit out BB with incomplete helices ✓
3. GNN, TNN, CNN, ANN frequencies

• Targets (5-10; 8) x Backbones (???) x Helices (≥500)=55,000
  • Backbones: similar, but not too similar to zif268; more than 1-2 aa changes, but <10
  • Helices fixed based on our program-- eventually saturates and levels out
    • Graph: # of var (# of tries by the computer) vs. % space covered

Proposed interactions between helical zinc finger residues and base pairs of the target DNA sequence (based on Persikov 2011 Persikov2011

Options for Target DNA Sequences / ZF Helices

1. F3(known) / F2(known) / F1(novel)
2. F3(known) / F2(SNP in b₁ position) / F1(known)
3. F3(unknown) / F2(unknown) / F2(unknown)

• Excluded Rare Codons (for E. coli)CodonUsage OpenWetWareCodonUsage NIHRareCodonCalculator:
  • CTA
  • ATA
  • CCC
  • CGA
  • CGG
  • AGA
  • AGG
  • GGA
  • GGG

References

<biblio>

1. Persikov2011 pmid=21572177
2. CodonUsage http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/in-vitro-genetics/codon-usage.html
3. OpenWetWareCodonUsage http://openwetware.org/wiki/Escherichia_coli/Codon_usage
4. NIHRareCodonCalculator http://nihserver.mbi.ucla.edu/RACC/

June 20th

• Grew up colony of the selection strain with pKD46 in an attempt to reach mid-log and create glycerol stock
  • Unable to reach mid-log, so going to leave growing over night and use saturated culture tomorrow
• Determined primers in order to piece together the omega subunit and ZFP genes into the pZE21G plasmid (spec cassette)
• Ran PCR on His3 locus and sent to GENEWIZ to be sequenced
  • used the same procedure as the earler WT/KO PCR, but with 1µL undiluted template and only His_F and His_R primers
  • ran 3 reactions and sent in three primers (His_F, His_R, His_internalR)

June 20th - Bioinformatics

Goals for the week

• Finish designing the chip, by Wednesday hopefully
  • Need chip order out, takes 4 weeks
  • Need all sequences by Friday!!!
• FIRST PRIORITY: If we can get Persikov to work, good!
  • Step one: get results he’s published, get the web app to "work" with his data, then OPEN data, and finally our data
• Brainstorming session (tomorrow?) to decide how many targets/sequences
• Determine the importance of the first/second/third nucleotide positions
  • Look at NGN, NTN, NAN, NCN (Not just GNN, etc.)
  • Pick a particular GNN, plot vs. TNN- is there a pronounced difference in position 1, or -1?

Today

• Testing Persikov's Data for validation
  • Persikov v. himself ✓
  • Persikov v. OPEN
  • Persikov v. our sequences

Probability data

• The following are graphs of the probability of finding each amino acid at each position on the alpha helix.

Probability data for the 783 fingers that bind to GNN triplets. Note the high probability of leucine at position 4 and arginine at position 6.	Probability data for the 128 fingers that bind to TNN triplets. Note the high probability of leucine at position 4.	Probability data for the 16 fingers that bind to CNN triplets. There may not be enough data to consider this information statistically significant	Probability data for the 29 fingers that bind to ANN triplets. There may not be enough data to consider this information statistically significant
Probability data for the 298 fingers that bind to NGN triplets. The position 4 leucine motif remains. There is also a high probability (> 0.5) of a histidine at position 3 and an arginine at position 6.	Probability data for the 177 fingers that bind to NTN triplets. The position 4 leucine motif remains.	Probability data for the 244 fingers that bind to NCN triplets. The position 4 leucine motif remains. There is also a very high probability of an arginine at position 6.	Probability data for the 248 fingers that bind to NAN triplets. The position 4 leucine motif remains. There is also a very high probability (> 0.75) of an asparagine at position 3 and an arginine at position 6.
Probability data for the 234 fingers that bind to NNG triplets. The position 4 leucine motif remains. There is also a very high probability (> 0.75) of an asparagine at position 1 and a high probability (> 0.5) of an aspartic acid at position 2 and an arginine at position 6.	Probability data for the 247 fingers that bind to NNT triplets. The position 4 leucine motif remains. There is also a high (> 0.5) probability of an arginine at position 6.	Probability data for the 262 fingers that bind to NNC triplets. The position 4 leucine motif remains. There is also a very high (> 0.75) probability of an arginine at position 6.	Probability data for the 218 fingers that bind to NNA triplets. The position 4 leucine motif remains. There is also a very high (> 0.75) probability of a glutamine at position -1 and an arginine at position 6.