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Template:Team:UC Berkeley/Notebook/CDB notes
From 2008.igem.org
6/4/08 - Today we continued training and discussed the project.
6/6/08 - Jin streaked a plate with MC1061 E. coli cells and incubated at 37 C.
6/7/08 - Plate moved to fridge.
6/8/08 - I chose an isolated colony from the plate and inoculated a few mL of fresh LB media with it. The tube was incubated on a shaker at 37 C for 24 hrs.
6/9/08 - Took 200 uL of saturated culture and added it to 8 mL of LB media at 10:45 AM. Grew for 2 hrs on a shaker at 37 C.
Split culture into 3 tubes with 2 mL each. Culture 1 - Control (no sound), grew in foam box. Culture 2 - Music, high volume for 1 hr. Culture 3 - Ultrasound bath for 1 hr.
Note - Didn't have lysozyme thawed for RNA purification. Will repeat experiment tomorrow.
Inoculated a new tube of media with an isolated MC1061 colony.
6/10/08 - Added 200 uL of saturated culture to 8 mL of LB media. Grew for 2 hrs on a shaker at 37 C.
Split culture into 3 tubes with 2 mL each. Culture 1 - Control (no sound), grew in foam box. Culture 2 - Music, high volume for 1/2 hr. Culture 3 - Ultrasound bath for 1/2 hr.
Performed RNA extraction with RNA protect kit for bacteria -
Protocol
Supplies Sterile, RNase-free pipet tips
Suitably sized tubes and microcentrifuge or centrifuge with appropriate rotors
Lysozyme (e.g., Sigma, cat. no. L7651) or appropriate lytic enzyme*
Tris and EDTA for preparing TE buffer
QIAGEN Proteinase K (see ordering information, page 47)
2 ml Safe-Lock tubes (Eppendorf, cat. no. 0030 120.094)*
14.3 M b-mercaptoethanol (b-ME) (stock solutions are usually 14.3 M)
RNeasy Protect Bacteria Mini Kit
Ethanol (96–100%), ethanol (80%), or ethanol (70%)†
Optional: RNase-Free DNase Set (see ordering information, page 47)
RNA zap
100 ml lysozyme-containing TE buffer (10 mg lysozyme in 25 ml RNase-free TE)
DEPC water
RNASE-free TE
beta mercaptoethanol
200 proof ethanol
RNase free needle and syringe
Before starting -
clean work area, gather all pipets and tips
mix Buffer RLT and beta mercaptoethanol (10 uL BME per 1 mL RLT) - need 20 uL BME in 2 mL RLT
mix TE Buffer with lysozyme (15 mg/mL) – need 7.5 mg lysosyme in 500 uL TE Buffer
dilute buffer RPE 1:4 with ethanol - need 44 mL ethanol
prepare DNase stock solution - dissolve solid DNase in 550 uL of RNase free water using an RNase free needle and syringe. Mix carefully with needle - DON'T centrifuge. Freeze single-use aliquots at -20C
After Sound Treatment
Protocol 4
in a 2 mL tube, add 1 mL RNA protect and 500 uL culture. Vortex 5 s and incubate 5 min (RT). Centrifuge for 10 min at 5000xg. Decant supernat. Dab inverted tube once on a paper towel - pellet may not be visible
Mix 15 uL Proteinase K with 100 uL TE Buffer. Add mixture to pellet and carefully resuspend the pellet by pipeting. Vortex for 10 s. Incubate for 10 min (RT) on a shaker (or vortex 10 s every 2 min) - extending incubation time may increase yield.
Add 350 uL of Buffer RLT and vortex vigorously- if there is particulate matter, centrifuge 2 min at > 8000 x g and use only supernatant
Add 200 uL of 96-100% ethanol and mix by pipetting (DON'T centrifuge)
Protocol 7
Transfer up to 700 uL of lysate, inc any ppt to RNeasy mini spin column in a 2 mL collection tube. Centrifuge for 15 s at max speed. Discard flow-through.
Repeat if there is remaining lysate
Appendix B - DNase digestion
Add 350 uL of Buffer RW1 to mini spin column. Centrifuge for 15 s at max speed. Discard flow-through
Add 10 uL of DNase 1 stock sln to 70 uL of Buffer RDD. Mix by inverting gently - don't vortex. Centrifuge briefly
Add DNase 1 mixture (80 uL) directly to mini spin column membrane and incubate (RT) for 15 min
Add 350 uL of Buffer RW1 to spin column. Wait 5 min, then centrifuge for 15 s at max speed. Discard flow through and collection tube
Protocol 7 cont.
Place spin column in a new 2 mL collection tube.
Add 500 uL of Buffer RPE to spin column. Centrifuge for 15 s at max speed. Discard flow-through
Add 500 uL of Buffer RPE. Centrifuge for 2 min at > 8000 x g. Carefully remove spin column from collection tube and discard tube and flow through.
optional - Place spin column in a new 2 ml tube and centrifuge for 1 min. Discard tube and flow through.
Place spin column in a new 1.5 mL collection tube.
Add 50 uL of RNase free water to spin column membrane. Centrifuge for 1 min at > 8000 x g to elute RNA
If expected yield is over 30 ug repeat last step. Add 30-50 uL of RNase free water to spin column membrane. Centrifuge for 1 min at > 8000 x g to elute RNA
Discard spin column and put tube on ice
Results
Sonic Bath - 5.1 ug/mL 260/280 - 1.61
Music - 1.4 ug/mL 260/280 - 1.72
Control - 4.2 ug/mL 260/280 - 1.67
Since purity is low, experiment was repeated with the following changes:
1) assumed a higher # of cells - increased TE Buffer to 200uL, Buffer RLT to 700 uL and ethanol to 500 uL.
2) centrifuged after addition of buffer RLT and used only supernatant.
3) eluted with 100 uL of RNase-free water (2 additions of 50 uL each) - Note - The concentration of RNA after the addition of 100 uL of water was substantially higher than the concentrations in any of the first samples, which were only eluted with 50 uL. Not sure why... but it's good because it means we would have enough total RNA for the microarray.
4) Centrifuged at max speed, except for the initial pellet.
Much better results this time 260/280 was 2.01 and the concentration was 20.6 ug/mL.
I inoculated some media and will try again tomorrow.
- Updated protocol here [http://docs.google.com/Doc?docid=ddh89z4j_11cnxq6jwr&hl=en]
6-11-08
Repeated updated protocol from 6/10/08
Results
Sonic Bath - 18.2 ug/mL 260/280 - 2.04
Music - 11.6 ug/mL 260/280 - 2.06
Control - 20.5 ug/mL 260/280 - 2.04
Used 10 uL to test the purity and concentration using a spectrophotometer. Don't have enough total RNA from the control sample for the microarray. Need to repeat control experiment to extract enough RNA. Need to concentrate the music and sonic bath samples to 0.5 ug/uL (use zymo clean-up reaction.)
6-12-08
Tried to concentrate sonic and music samples using a Zymo DNA kit. Failed miserably! Both purity and concentration dropped. Need an RNA zymo kit.
Repeated experiment for all three conditions. Repeated protocol from 6-10-08, except used 150 uL collected in 3 sample tubes for each condition.
Spectrophotometer results here [http://spreadsheets.google.com/ccc?key=pWg-yF6Hhkf_1ytn5MB1sWA&hl=en]
Tomorrow, will concentrate RNA and run on a gel.
6-13-08
Used the microcon centrifugal filter devices ultracel YM-10 to concentrate RNA. Used 100 uL of sound sample and 150 uL of control and ultrasound sample. (Note: 1 of the 3 sound samples initially showed a low purity and was not used, hence only 100 uL of this sample). Used 50 uL of RNase free water to clear glycerol from tube before use. Spun at 14000 xg for 9 minutes (8 minutes for the sound sample). Flip over tube and run for 3 min at 1000xg to elute. The volume was reduced to approximately 60 uL of each sample. All samples show a 260/280 ratio of 2.01-2.02 (pure).
Final concentrations and purity
Sonic Bath - 0.735 ug/uL 260/280 - 2.02
Music - 0.8 ug/uL 260/280 - 2.01
Control - 0.52 ug/uL 260/280 - 2.01
Loaded 1 ug of each sample and ran a 1.2% e-gel. Two distinct bands appear for each sample (23S and 16S subunits).
6-16-08
Submitted form, 3 samples and 3 affymetrx microarray chips to Justin at 255 LSA. He will contact Chris when chips have been analyzed.
6-23-08
Didn't have much to do last week. Helped the others with gels, digestion, mini-prep and mixing buffers. Did some research on holin-antiholin. Downloaded GCOS (Gene chip operating software)- freeware from affemetrix to analyze microarrays. Played with some sample data so that I'm ready for microarray analysis when the time comes.
There was a delay with the FGL, so I won't get data for a few more days. Trying to keep myself from yelling at Justin (the microarray guy) for not telling us about the delay until today.
Began researching papers to create a model for the kinetics of the holin-antiholin-lysozyme system. Will model using a system of linear equations. It will be a more abstract model (not based on experimental data, which would be hard to measure). Need a system of equations for each of three conditions (before depolarization, after depolarization, after enough holes form to allow lysozyme to cross IM).
6-27-08
Still no microarray data. Justin from FGL hasn't ordered the reagents yet. Met with Karen Vranizan, the stats woman from FGL. She advised me not to use the GCOS program because there is no easy way to normalize samples across multiple chips. She recommended RMA Express, a freeware program that will normalize our data. I downloaded the program and began to learn how to use it so when I actually get data, I can analyze it.
I created construction files for three ultrasound-responsive promoters for the grpE, recA, fabA genes based on primers from the following papers
recA - primers for RecA promoter from Vollmer et al., 1997. [http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=168554&blobtype=pdf]
fabA - Primers for fabA proteins from Bocher, et. al 2002
[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T3C-44W2K4N-6&_user=4420&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000059607&_version=1&_urlVersion=0&_userid=4420&md5=b221542b03abe204b6638860617ae471]
Want to make a fourth promoter for katG, but it has four internal restriction sites, so I'm not sure if its worth ordering ten oligos. Also, I don't know how to get make a mutation "silent" in a noncoding region. Will ask for Jin's advice when he comes in.
Update - not making a basic part for katG promoter (too many internal sites). Updated construction file on wiki and google docs.
7-1-08
Received oligos for greP, recA and fabB genes. Did PCR, ligation, transformation and plated on spec plates. Will incubate overnight.
Created files for two prepro (signal peptide) sequences from phoA and lamB genes.
Signal peptide (prepro) sequences for phoA gene – MKQSTIALALLPLLFTPVTKA obtained from Musial-Siwek, et al, 2005 [http://pubs.acs.org/cgi-bin/article.cgi/bichaw/2005/44/i42/pdf/bi050882k.pdf] and Wang, 2004 [http://pubs.acs.org/cgi-bin/article.cgi/bichaw/2004/43/i41/pdf/bi049485k.pdf]
Signal peptide (prepro) sequences for lamB gene– MMITLRKLPLAVAVAAGVMSAQAMA obtained from Jones and Gierasch, 1994 [http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1225516&blobtype=pdf]
Will present kinetics model to group tomorrow at 2pm.
7-4-08
Well, it's the fourth of july and I went to lab for an hour to check on some samples. Ended up staying til 10 pm. Lab just has a way of sucking you in (it's like being in a casino).
7-10-08
Began mini-prepping 200 samples by hand in individual tubes. Dr. Anderson is worried about tube switching, so we're going to use the magnetic bead assembly instead.
This week, I've been updating and combining our spreadsheets from google docs. We had another modeling meeting to discuss how best to model holin/antiholin/lysozyme interactions. Uploaded some papers onto google groups. Terry presented some good papers that I still need to read.
I finally got my microarray data. Will analyze this afternoon to see if I can find some genes that are upregulated in response to sound, relative to the control. If there are no genes that are significantly upregulated, I'll look at the ultrasound chip.
7-14-08
Have been trying to analyze our microarray data, however our control chip has a spot that is making part of the chip appear brighter than it actually is. According to Affymetrix, the spot is an area of the chip that was improperly cleaned before hybridization. I haven't yet figured out how to exclude the probes from this section. Dr. Anderson has suggested we outsource the data analysis. I will be researching industry analysis companies. I'm also looking for a local microarray processing facility to process the last three chips since the work of the core facility was slow and sloppy.
Began to make composite parts for our promoters bolA, ftsQ, ftsAZ, Phns, pspv2, pspv, PfhuA, rrnb-p1, pdps, ptet, grpE recA, fabA, pBad-spvR-spv2, spv-spv2 and ptet-spvR-spv2, using the RFP gene in pBCA1107 plasmid.
7-15-08
Some of the promoters-RFP constructs did not grow properly and will need to be remade. Also the DNA from the smaller promoters was too dilute to use Clone Wells. Will need to repeat experiment and cut the DNA out from an EtBr gel.
Ordered oligos for the promoters of the cadA and cadB genes, which are upregulated greatly in response to sound and US. (pathway: low pH or high lysine)
Primers for cadAB genes from Kuper and Jung, 2005 [http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowPDF&ArtikelNr=90346&Ausgabe=231585&ProduktNr=228391&filename=90346.pdf] , [http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=205747&blobtype=pdf]
7-16-08
Cloned the Beta-gal (lacZ gene) and will use it to test promoters using assembly vectors. Looking for a non-core facility for microarray hybridization. Most of the ones I've found so far only process human and mouse chips. Will keep looking.
7-18-08
Made an assembly tree for the lacZ, promoter and rbs genes. Need to put an rbs on the lacZ gene and then make composite parts with promoter.rbs1-A.lacZ. Took a look at the beta-galacotosidase assay. Shouldn't be too difficult.
7-20-08
Made too many stupid mistakes today. Ran my gel too long and lost my bands. Have to repeat the digestion mapping. I need to slow down and not try to do too many things at once.
7-21-08
Making composite parts with promoter.rbs1-A.lacZ. Have bolA, ftsAZ, Phns, pspv2, PfhuA and fabA. Digestion mapping revealed that ftsQ and pdps are wrong. Growing GrpE, recA, ptet, pCad, lacZ and pspv from other assembly vectors [http://spreadsheets.google.com/ccc?key=pWg-yF6Hhkf-ZSC-bGe7pMw&hl=en]. Testing assembly vectors using LacZ insert.
7-22-08
Assembly vectors didn't grow. Not sure why, but lacZ insert also didn't grow in a tested assembly vector. Will start over tomorrow with inserts that I know are correct. mini-prepped, digested and transformed inserts from yesterday into AC assembly vectors (except lacZ which went into AK). Ordered oligos for the cadC gene.
7-23-08
Chris wants us to use GFP to measure strength of promoters so I have abandoned the composite part assembly. Obtained plasmid DNA from a GFP vector (pBca1256-bsl-1007). Will amplify tonight.
7-24-08
At mini-meeting, we talked about modeling. I will start working on the transfer function for next weeks modling meeting.
We decided to go with the Gladstone UCSF core facility for microarray processing. Paul Lum is helping me set up a focused US experiment so that I can use a higher frequency, focused US for the next microarray experiment.
GFP amplification went well. Moved pdpns, ptet, pCadAB, pspv, rrnb-p1, grpE and recA into AC plasmid and transformed into lefty cells. Used AC plasmid mini-preps to move bolA, ftsAZ, phns, pspv2, phfu and fabA into lefty cells. Transfered GFP part into CK and transformed into righty cells. Transfered lacZ into AK and transformed into righty cells.
Tested vectors using Molly's basic parts.
PCR-ed oligos for ygeF and cadC gene promoters.
7-25-08
Vectors did not grow (except AK (20) and KA (1)). I assume they are no good or way too dilute since this is the second test of the vectors.
All promoter transfers, GFP and LacZ grew well (except pCadAB). Transfered 3 cultures of each of the transfers to liquid media. Will mini-prep and map tomorrow. For some reason, pCadAB did not grow. Today I digested pCadAB from mini-prep of basic part and from pCadAB-AK. Will try to transfer tonight. Also will digest, ligate and transform cadC, ygeF into AC plasmid and lefty cell. Also digesting and transforming one of Jin's sample (TtRF) in KC and righty cells.
Sent partially completed UCSF microarray app to Chris.
Found a florescence plate reader at Brad's lab on Potter street, but will look for one here on campus.
7-26-08
Mini-prepped yesterdays vectors and sent in for sequencing. Picked colonies and grew cadC, cadAB, ygeF and TtRf samples. Once I have sequencing data, I will start 1-2-3 assembly.
Worked on transfer function for model. I have a basic transfer function done. It's a piecewise function and I think it has to be because the behavior is radically different after the loss of PMF. There are some assumptions in the transfer function that I must justify.
7-27-08
Took a look at sequencing files. Most look good and I will start assembly on those tomorrow. Need to re-do rrnb-P1. Don't know why I'm having trouble with this part. It's just a promoter, not toxic or anything. Bing is also having trouble with it.
Have a meeting with Paul Lum tomorrow at 10 to talk about ultrasound.
7-28-08
Worked on setting up the transducer assembly. This way we can deliver a much higher frequency of ultrasound and better quantify our experiment. I'm still kind of sketchy on the working of the oscillator.
Used 1-2-3 assembly on all promoters, except cadAB, cadC, ygeF and ftzQ which are being sequenced. However, I made a terrible mistake and used NEB buffer 2 without ATP. I tried adding ATP during the ligase step, but I'm not sure if it will work. Also added REs to my GFP miniprep, but I discovered the mistake quickly and I think I zymo-ed in time. We'll see what grows tonight.
7-29-08
Only one of the 1-2-3 assemblies worked (pspv2). I am redoing them today. All of the sequencing for the new promoters looks ok, except for cadC. Both samples were bad reads, so Richard is redoing them for me. trFA looks good, so I gave it to Madhvi for transfer.
Terry is helping me set-up the ultrasound apparatus this afternoon.
7-30-08
I finally have a decent understanding of how the oscilliscope and transducer assembly work. I'd like to hook the set-up to a power amplifier to boost signal intensity. I need to talk to Paul about it though because I don't want to fry his transducer.
Re-did the promoter.GFP assembly only to discover that there is something wrong with my GFP part. Chris wants to change the assembly again. He gave me a GFP plasmid in the entry vector pBca1256-Bsl1007. I'm cutting it with Bgl/Eco and cutting the promoters with eco/bam and ligating them together. I'm using lefty cells for the promoters and righty cells for the GFP plasmid so that I can add Bam and BglII to the ligation mixture to eliminate background. It involves more gel purifying, but less time.
8-2-08
Still moving promoters into GFP plasmid. Some of the smaller promoters must be cut with different enzymes because they're too small to resolve on a gel. I also worked on modeling. Fun times on a beautiful saturday afternoon that's quickly becoming evening.
8-4-08
Yesterday, I came in briefly to pick colonies and incubated overnight. My gels shows that fhuA, recA, dps, cadAB, ftsQ, ftsAZ, ygeF, pspv, pspv2, hns, grpE are the correct size. rrnb-p1, fabA and ptet are wrong. Still need to clone cadC into assembly vector.
8-5-08
Tried to remake ptet.GFP and rrnb-P1.GFP and fabA.GFP. However the cells did not grow. Not sure what I did wrong though. Will have to repeat.
8-6-08
Started adding parts to the registry. Amplified GFP righty, cadC lefty. Set up sound promoter tests. Digest ptet with Xho/Bam because its too small to gel purify when cut with Eco/Bam.
Trying to find a way to get more power from the ultrasound transducer set-up. Paul has a "big bang" box that can provide a lot more power than the transmitter/pulser that I've been using.
8-8-08
need to sequence cadAB, grpE.GFP. Test growth phase promoters. Very rough test, just grew cultures to saturation, inoculated fresh LB media and grew to midlog. Spun down cells and looked for expression of GFP. Phns and pspv are on at both saturation and midlog. bolA, ftsAZ, ftsQ, pspv, pdps and pspv2 are off at both saturation and midlog. pfhuA appears to be on at midlog, but off at saturation? This puzzles me because the GFP protein takes time to breakdown. I'll repeat this experiment to confirm my results.
8-9-08
ygeF, bolA, cadAB and recA show no response to low frequency ultrasound. ygeF and cadAB show no response to audible sound either. None of the promoters responded to high frequency, high intensity ultrasound. grpE appears to be our best candidate. It is expressed more strongly at 37C than at 25C or 30C. There was no growth at 43C.
8-11-08
I think I've found it! A gene that responds to ultrasound. Need to reconfirm with better controls.
8-13-08
Sequencing shows that my "sound" promoter is actually phfuA, an iron dependent promoter. I grew all the promoters on the same plate and I must have inadvertently chosen phfuA instead of fabA.
8-18-08
We don't have a sound promoter. I've seen far too many negative controls turn on to trust any of the promoters. grpE our best hope, was too strong of a constitutive promoter to use as an inducible promoter.
