"
E. cholerex
From 2012.igem.org
February 16, 2012
We did PCR for the amylase gene. The protocol is as follows:
-35 ul ddH2O -10 ul 5X Phusion buffer -1.5 ul 10mM dNTP's -1 ul each primer (BI124 and BI123) -1 ul diluted template E. coli -0.5 ul Phusion Polymerase
-AB
February 23, 2012
Amylase PCR product was run out on gel. PCR was unsuccessful; it is possible that the reverse primer was doubled and the forward primer was left out. We redid the PCR using the same protocol. -AB
February 24, 2012
We did PCR of the Holin gene and ran the gel to check the PCR products from 2/23/12. The PCR of amylase was again unsuccessful. It is possible that it didn't work because the PCR machine was set for Taq polymerase cycles rather than Phusion. The protocol for Holin PCR is as follows:
-35 ul ddH2O -10 ul 5X Phusion buffer -1.5 ul 10mM dNTP's -1 ul each primer (BI129 and BI130) -4 ul T4 bacteriophage template (obtained from Dr. Breakwell's lab) -0.5 ul Phusion Polymerase
AB
March 2, 2012
We ran a gel with all of our PCR products. The image of the results are found in lab notebook AB, page 14. PCR products included are: Control, Cre, Lox, 406, Amylase, GBP, QBP, Holin (from bacteria-containing template), and Holin, respectively. -AB
March 6, 2012
PCR of Holin didn't work again. We believed there may have been a problem with the template. We plated E. coli and dropped a few ul's of T4 phage to develop plaques overnight. -AB
March 8, 2012
We did PCR of Holin again. This time we used T4 bacteriophage taken from plaques set up on 3/6/12. Standard protocol was used. -AB
March 9, 2012
We performed low melt gel of the the Restriction Digests. The order of the digests are as follows;
1). Ladder 2).GBP 3). PBAD PST1 Xhol 4). (J) Plat ECOR1 Pst1 5). (Y) Plat ECOR1 Pst1 6). QBP 7). Ladder 8). LOC PCR 9). LOC Plasmid 10). Empty 11). Empty 12). Empty
~JT~
March 13, 2012
We ran the gel for our Holin PCR product and a control. The image of the results is in notebook AB, page 16. The PCR was unsuccessful. The control and our Holin product appeared to be the same size. We believe we were seeing the primers in the gel. -AB
March 15, 2012
Today we did the transformation (KSB-32) -Thaw DH5 alpha cells on ice -Melt ligations at 65 C . -Have 42 C heatblock ready and LB plates with antibiotic ready -When cells thawed, at 2 microliters of ligation mix to about 25 microliters cells . -Vortex and put on ice 5-10 minutes -Heat shock at 42 C for 60 seconds -Immediately place tubes on ice 2-5 minutes - Add .5 mL plain LB . -incubate at 37 C for 30 minutes (plates had ampicillin) -Plated 100 microliters cells onto plates (three of the good ligation (J), one of the possibly wrong ligation (Y), and one control for each)
We also started a liquid culture of T4 phage using 10 ul of T4 and E. coli. -AB
March 16, 2012
-We found growth on the incubated transformations; Trans J, Trans Y and Trans Y Control.
-We picked 8 colonies from the Trans J plates, and added them separately to tubes containing 50ul of ddH20.
-We streaked the 8 solutions onto plates and incubated at 30 C.
~JT~
-We did colony PCR of the 8 transformed colonies using 23 microliters of a master mix and 2 microliters of each colony template and a control of 23 microliters of master mix.-KJ
In addition, we did PCR of Holin using T4 phage from the liquid culture started on 3/15/12. We used standard protocol, with 4 ul of the T4 bacteriophage template. It was placed in the thermocycler using the high fusion program. -AB
March 22, 2012
Ran gels for remaining PCR products (Holin, Dnase, Holin control, C (Q), C (CH), Cre, thermosensor 1-8). Standard protocol. Image is on 3/22-AB page 18. -AB
March 27, 2012
Tube labeling Nomenclature -Primers BI#[[ ]]
-PCR Clean Up PCR UP DATE [[INITIALS]
-Restriction Digest RD DATE [[INITIALS]
-Gel slice from Lowmelt GS DATE [[INITIALS]
-Ligation Lig DATE [[INITIALS]
Primers for NucB, DNase1, Aiia,
work in progress ~JT
Ran PCR for Dnase, NucB and Aiia. Standard protocol. -AB
March 29, 2012
Ran gel for PCR products from 3/27. Standard protocol. Image is on 3/29-AB page 19. The gel image also contains results for the thermosensor group (Lox, 1-8). -AB
April 24, 2012
We did PCR for NucB, DNase1, and Aiia. We used standard protocol,which involves the phusion. (see entry.....)
The templates were as follows:
NucB - Bacilus. Subtilus, strain 11774. (labeled BS 11774)
Aiia - Bacilus. Subtilus, strain 11774. (labeled BS 11774)
DNase1 - Homosapien. Obtained from and isolated by Britt German. (Labeled BG DNA)
We boiled a colony of Bacilus. Subtilus, strain 11774 in 50 ul of water for 5minutes to use as the template. We then put the specimens in the PCR machine.
~JT~
I began searching cholera quorum sensing genes on GenBank and found hits for protein sequences for LuxP, LuxQ and LuxU- KJ
~YC~ Redid the holin primers and ordered them.
We ran the restriction digest of insert and vector for Amylase and CytR that had been purified. Standard protocol, with both digests requiring the restriction enzymes EcoR1 and HindIII. The digests were placed in the 37 degree incubator overnight. -AB
Research assignments:
-Quorum sensing: Joe, Kyle
-Flagellar movement: Amy
-Kill switch: Yuri, Kellie
-Toxins: Audrey, Kellie
April 25, 2012
We collected the PCR products, and loaded them (NucB, DNase1, Aiia) with their forward and reverse primers respectively. We also loaded a control for each of them. We ran them out on a gel for 22 minutes. There were 6 product run in total. The order of products is as follows;
1). Ladder 2). 5-4A (QBP) 3). DNase1 4). DNase1 control 5). NucB 6). NucB control 7). Aiia 8). Aiia control
Results were positive for NucB and DNase1. We will try the Aiia again with a different template. ~JT~
The digests were removed from the incubator and placed in the fridge. -AB
April 27, 2012
The Digests were loaded and ran out on the Low Melt Gel [90V for 52 Minutes ~40-50uL of digest loaded in each well, 10uL for ladder]. Used left over from Julie's run of the plasmids on the 24th. Loading: 1)Ladder 2)Amylase Digest 3)CytR Digest 4)pLat Vector Digest. Ladder well had a leak and thus did not run. Obtained bands from CytR(well 3) and Amylase(well 2). Well 2 band was very thick and clear, managed to cut a lot of gel off and although there was no ladder there were no other bands visible. Well 3 band was clear yet it was not very thick and I couldn't cut off a lot of gel as it was such a faint band. -AF
We boiled three strains of Bacilus subtilus in preparation of making three more templates for Aiia. We used these three strains to redo a Phusion PCR (protocol above on Feb 16) with an elongation time of 3 minutes for AiiA. ~JT~ KJ
Today, we also did the purification of nucb and dnase I PCR product. We followed the Qiagen kit protocol. The purified PCR products are frozen down in slot 1H and 1I. We still need to run the gel, but the gel has been made and we will run it on Monday. -KB
April 30th, 2012
A Nano-drop was performed for the Purified Plasmids 1A-E.
1: 1.92 (260/280) 135.5 (ng/uL)
3: 1.92 (260/280) 179.1 (ng/uL)
4: 1.93 (260/280) 184.7 (ng/uL)
5: 1.93 (260/280) 145.4 (ng/uL)
6: 1.90 (260/280) 137.8 (ng/uL)
We loaded water onto the nano-drop to clean. Next we loaded Sigma Elusion Buffer to blank the system.
Restriction Digests were performed for the Purified Plasmids 1A-E using #2 Buffer, and Restriction enzymes for EcoR1 and HindIII to enable us to insert both Amylase and CytR which both contain EcoR1 and HindIII. -AF
We ran a gel for the Aiia PCR products. We also ran a gel of the purified NucB and DNaseI genes.~ YC
I researched the flagellar motor. For the links to papers look at the google doc. -AB
May 1, 2012
Today we did the restriction digest of our vectors using the standard protocol. -KB
NucB (EcoRI-XhoI)
DNaseI (EcoRI-XhoI)
pLAT w/ signal (EcoRI-XhoI)
We ran a low melt gel of all the inserts and vectors to prep them for ligation. ~YC
Low melt gel:
Top
Lane 1: Ladder
Lane 2: Cholera Restriction Digest 1
Lane 3: Cholera Restriction Digest 3
Lane 4: Cholera Restriction Digest 4
Lane 5: Cholera Restriction Digest 5
Lane 6: Cholera Restriction Digest 6
These restriction digests use restriction enzymes EcoRI and Hind III
Lane 9: Malaria's 5-2a
Lane 10: Malaria's 6-1
Bottom
Lane 1: Ladder
Lane 2: Cholera Restriction Digest 1
Lane 3: Cholera Restriction Digest 3
Lane 4: Cholera Restriction Digest 4
Lane 5: Cholera Restriction Digest 5
Lane 6: Cholera Restriction Digest 6
Lane 7: NucB digest
Lane 8: DNase I digest
These restriction digests use restriction enzymes EcoRI and XhoI
-KB
Ran PCR for Anti-LPS with E. coli template. Standard Phusion protocol using Phusion program on PCR machine. -AB
NucB digest failed. ~YC
May 4, 2012
We did the ligation for Dnase, CytR and Amylase using the low-melt gel products obtained on 5/3. Standard protocol. Ligations were left on a tube rack to incubate at room temperature. -AB
We researched, crossed referenced and blasted the genes until we found a compatible strain of Vibrio cholerae that we are planning on using for our different gene insertions. The ones we currently are interested in are;
Fig 1.) 26 2.) 27
NCTC Number 1.) 8021 2.) 7254
Type of strain 1.) Vibrio choleraeasiaticae (V. comma) T.Sp 2.) Vibrio choleraeasiaticae (V. comma) T.Sp / TWT
-V. cholerae IEC224 chromosome 1
-V. cholerae 01 st. 2010EL-1786
-V. cholerae LMA3894-4
-V. cholerae 0395
So far the 0395 strain has had the best results when blasted, showing recombinant of 98-100% for most.
We will continue to research.
~JT~ ~KJ~
May 7, 2012
Ran gel for Anti-LPS PCR product from 5/1. -AB Did PCR of NucB.
May 8, 2012
Ran a gel of the Anti-LPS and NucB PCR products.
Did PCR of Holin. We followed standard protocol from the lab protocol sheet.
~AF~JT~
Redid PCR of Anti-LPS.
Did PCR cleanup NucB PCR product from 5/7.
Did Restriction digest on pLAT with pBAD and on the purified NucB.
~YC~
May 11, 2012
Ran gel for Holin and Anti-LPS but neither PCR's were successful. -AB
Ran a low melt gel of the digested plasmid (pLAT with pBAD). ~YC
May 14, 2012
Annealed two strands of the signal sequence, preparing it for the ligation.
May 15, 2012
Preliminary designs for our Quorum/Flagellum hybrid was discussed throughout class:
Trg (from Ecoli K-12):
0-272: Sensing Domain (226-273: Hamp) 273+ Signaling Domain a.k.a What we want
Cqss (Cholerea O3995):
0-183: Ligand Binding 184-250: HisK (Dimerizes)314+ Phosphotase
Start: Cqss 1-183 / 1-252 / 1-314 End: Trg 226-end / 273-end
All in all we will be designing 6 different systems
Audrey and I designed the primers HapR and LuxO for quorum sensing, they need to be verified before ordering. A plate was made to create T4 plaques, after three hours plaques were seen, yet after being left overnight in the 30 degree incubate the plate failed. ~AF
Kellie and i performed DNA extraction from an apple using the DNA extraction kit. This was to create a template for the apple flavanoid protein. The extraction failed.
~JT~
May 16, 2012
Ran PCR for Apple flavinoid. -AB
May 18, 2012
We extracted DNA from apple using dirty protocol. We ground apple down, until it was a fine paste. Afterwards we added desk swabbing solution, allowing cells to break up. We then collected the juice by filtering the paste into a test tube through filter paper. The test tubes were then chilled in the refrigerator. ~JT~
Ran gel for apple flavinoid PCR product. -AB
Re did a plain LB plate of Ecoli with T4 drops in the hopes of developing plaques. incubated at 37 degrees ~AF
May 21, 2012
Getting DNA for Apple flavinoid (Dirty Procedure):
Once chilled alcohol is poured on the top layer of chilled apple juice mix, we waited for DNA to raise to the top. Once DNA was visible it was sucked up with a pipette, deposited into eppendorfs and centrifuged down. Supernate was disposed of and then the pellets at the bottom were re-suspended in 100uL of ddH2O and then boiled in PCR machine. ~AF
May 22, 2012
We prepared and carried out PCR for the apple flavanoid, using the templates prepared yesterday.
We followed PCR protocol, using primers 117 and 118. We did four samples, with one control.
~JT~
Kyle, Dr. Grose and I made primers for the chimeric proteins.
~YC~
Amy and I did the transformations for the ligations of the PLat with a signal. We followed the protocol given in the sheet. After plating the Plat we put them in the 37 degree incubation room over night.
~JT~
Ran gel for Apple Flavanoid. Image and order of gel wells are on AB-5/22 (page 24). -AB
May 25, 2012
Kyle and I did the colony PCR for the pLAT+sigal plasmid ~YC
May 29, 2012
The PCR products of the signal plasmids that were in the incubation at 37 degrees were left too long, and dried out. We rehydrated them, and re-plated them. We also reselected colonies from the original Plat plate, and plated them out. We put both plates in the incubation room at 37 degree. -After 2 hours growth has begun.
~JT~
We ran the signal sequence plasmid colony PCR on gel and it failed miserably. So today we redid the colony PCR under the direct supervision of Jordan.
-KJ-
May 30, 2012
Ran gel for colony PCR's 1-8. Image and gel well order AB-5/30. -AB
June 1, 2012
My brother's birthday!!!! (that is Yuri's brother's birthday)
PCR started for all 10 of the chimeric proteins we have designed. We have yet to get our hands on the elusive V.cholerae sample from Dr. Robison's lab. Hopefully we will get that soon. Some of our PCRs seem to have worked, but we need to double check against the ladder (as soon as we know what the ladder is showing, because I've no idea what the sizes are).
pLAT digested in preparation for the insertion of the medium promoter that will be used for the chimeric proteins. A low melt gel electrophoresis was ran, and the bands were cut out.
Promoter strands were annealed, and are ready for insertion into pLAT. Yuri was assisted in the gel prep and running by Joe.
~brought to you by Yuri the Wise, and Joseph I King of the Romans~
June 4, 2012
We started overnights on plasmids 2, 5, and 7 for plasmid purification. These plasmids should, according to our colony PCR results, have the signal insert. -KB
Overnights were made by using a sterile method, we poured LB into tubes and then used a pipette with a new tip to pick up a colony from each (re hydrated plate) streak of the Plasmids. After a colony was picked up in was then shot into an overnight tube, labeled, and placed into the shaker in the 37degree incubator. ~AF
June 5, 2012
KB performed the plasmid preps on 2, 5, and 7 from our overnights started yesterday. The plasmid prep kit was used. -KB
PCR of Phlorectin (Apple) was re done. The template we used was the product of the first PCR we did, this first PCR worked but was extreemly faint so we decided to use it as template to re-amplify and hopefully get a bright band. 1-4 of the previous PCR were used but #3 of the following may or may not have worked, it was not verified. ~AF,JT
Cholera plate was collected from Dr. Robison And gram stain performed to verify it was cholera. Pink, slightly bent, rod-shaped bacteria was observed. ~AF,JT
Purified plasmid (pLAT + signal) 2, 5, and 7 have been prepared for sequencing. The tubes were labled pLS 2, pLS 5, and pLS 7. 2 microliters of plasmid was mixed with 1 microliter of primer IG57. -KB
We set up overnights of K12 E. coli for the a redo of T4 plaques and of V. cholerae for biofilm formation tomorrow. We also prepared a 0.1% crystal violet solution for tomorrow's biofilm formation methods. -AB
Ligation was performed on the Plat plasmid with a medium promoter. Protocol was followed from the protocol sheet. A control was made with it, and the insert was omitted. They have both been incubated at room temperature for 30 minutes. ~JT~
0.1% solution of Powdered Crystal Violet to ddH20 was prepared: 10mL of H20 was mixed with 1g of powder and was stored in the refrigerator. An overnight of Cholera was made by shooting a pipette tip of a cholera colony into 5mL plain LB and put in 37 degree incubator shaker for Biofilm prep tomorrow ~ AF, AB
Corrected three primers for the chimeric proteins BI 213, BI 204, and BI206 ~Yuri the Great~
June 6, 2012
Ran PCR for DNAse from B.subtilis. The templates used were from the plates in the fridge labeled 1-48, 11774, 6033. PCR tubes were labeled as BDNAse
C - control
6 - from 6033 template
1-48 - from 1-48 template
1 - from 11774 template
Prepared a new gel for running electrophoresis of our PCR products. ~Yuri the Terrible~
We prepared 3 tubes for biofilm formation. The control contained 5mL LB. The two remaining tubes contained 5mL LB and 50uL of cholera liquid culture that was prepared 6/5/12. The test tubes were placed in the 37 degree incubator for 24+ hours. Also, a lawn of E. coli with 10uL spots of T4 was plated and placed in the 37 degree incubator for plaques. -AB
June 8, 2012
We DNA purified the PCR products from the first step of the chimeric proteins.
These are as follows; A, AQ(repeat of A; A naught), C, D, F. They are in the freezer awaiting for the next step. ~JT~
Ran a gel for the DNAse from B.subtilis. The PCR from the plate labeled 11774 worked.
We re-ran PCRs for the chimeric proteins B, H, Z9, and Z10. ~Yuri of Silla~
June 11, 2012
We set up a PCR for the chimeric proteins using cholera as the template. -AB, JT, YC
We set up the restriction digests on the plasmid with signal sequence. We set up three with EcoRI and Xho I for the insertion of DNase I, NucB, and AiiA. We also set up two with EcoRI and HindIII for Amylase and Cyt R. One for savinase needs to be done when we have more plasmid with signal sequence. -KB and AF
T-4 Phage SUCK!!!!!
After the realization that all of chimeric proteins were messed up and therefore useless, we had to start anew on our path to creating the protein to let us direct cholera towards the cholera biofilms. Because we are missing primers for two of our constructs, we only were able to restart on eight of the proteins 6 LuxN-Trg (LutR?) and the 2 LuxN-EnvZ (LuxNZ?).
The following labeling system has been used and will continue to be used for our constructs until further notification.
A - LuxN-Trg (made with primer BI200)
B - LuxN-Trg (BI201)
C - LuxN-Trg (BI202)
D - LuxN-Trg (BI203)
E - LuxN-Trg (BI204)
F - LuxN-Trg (BI205)
G - LuxN-Trg (BI206)
H - LuxN-Trg (BI207)
I - LuxN-Trg (BI213)
Z9 - LuxN-EnvZ (BI209)
Z10- LuxN-EnvZ (BI210)
Despite this minor setback caused by our friendly Chilean, we are now on our way to success!
~YC~JT~
June 18, 2012
We started growing up the plaques for the T4 phage. They are in the 37 incubator. Let's get that out tomorrow! -KH (formerly KB)
Low melt gel is ready to load and run on restriction digests. Gel setup and run for chimeric PCR products. The order on the gel is alphabetical order (A, A control, B B control..... Z9 Z9 control, etc.) -AB, JT, KJ and KBH
June 19, 2012
T4 phage did not grow. We ran low melt gel on the restriction digests of the plamid with signal sequence. Gel did not work, so we need to grow up more plasmid since we lost all those things. Peace. -KJ, AB, JT and KH
Took a picture of our gel to check chimeric sowing PCR products and all failed except for A, Z9 and Z10. Double checking primers.-KJ
AB, the PCR goddess, with the assistance of JT and KH, started the Holin PCR using the IGEM part as a template. May this work better than growing T4 phage. -KH
We set up the restriction digest for DNase from Bacillus subtillus using XhoI and EcoRI and intend to leave the digest in the 37 incubator until tomorrow. -KH
June 21, 2012
JT and KBH started the overnight from the colony PCR of plamid 7 (the plasmid 2 was not still on the plate, but those are the two plasmids that sequencing worked on). We can do a plasmid prep and then restriction digest on that next. -KH
Ran a gel for the Holin PCR products. -AB
We re-did the first step of the chimeric sowing PCR under the expert direct supervision of Jordan with B(BI201), C(BI202), D (BI203), E (BI204), F(BI205), G (BI206), H (BI207) and I (BI207). -KJ, JT, AB
We did DNA purification of A, Z9 and Z10, chimeric proteins that worked for the first step of the sowing PCR- KJ, AF
June 22, 2012
Ran Gel for Chimeric Protein PCRs and Holin
Row 1: ladder, B, BC, C, CC, D, DC, E, EC, F, FC, G, GC
Row 2: H, HC, I, IC, Holin, Holin C
~AB
June 25, 2012
Made 2 overnights each of Colonies 2 and 7 for plasmid preps. Split team into group prjects:
Natural System: Amy, Kellie, & Joe
Bio Busters: Audrey, Joe T., and Justin
QrrS/EnvZ: Kyle & Yuri
Alt. Pathway to be inserted into Ecoli: sensing = Repression of LuxO > Hap R > QFP
Non sensing = LuxO > Micro RNA 1 > RFP
~AF
Also, we performed Restriction Digests on the plasmid with signal sequence for insertion of DNaseI, and NucB using EcoRI and XhoI, Amylase and CytR using EcoRI and HinDIII, and B. subtillus DNase using BamHI and EcoRI.
We also did more plasmid preps.
-KH
Step 1 PCR for remaining chimeric proteins were successful, holin failed again. Did DNA purification of chimeric proteins. During last step, the centrifuge snapped off the lids of B, C, and D. DNA was still obtained but will need to be sequenced to separate them from each other. Step 2 of sowing PCR was performed with all chimeric proteins. -KJ, JT, AB
June 26, 2012
We did a plasmid prep on the the overnights from yesterday. We have two done for plasmid 7 with signal sequence and one done for plasmid 2 with signal sequence. -KH
We plated out samples of the plasmid 7 and plasmid 2 to have more colonies in stock and ready for more PCR/testing etc. We left them to incubate in the 37degree room.
~JT, AF~
We made a gel and ran our PCR products from step 2 of the sewing PCR. It failed and only had faint bands. We reviewed the primers and genes and Kyle discovered we were using the wrong reverse primers. We redid the PCR with the correct primers. ~JT, KJ~
We made a low-melt gel and ran the digest products from yesterday. These contained the plasmid with signal sequence. The low-melt gel products are now in tubes labeled with the name of each biofilm degrading enzyme and BB. -AB
June 27, 2012
We planned out our 96 well plates with Dr. Grose and set up the next few weeks of work.
We loaded the wells for a gel of the sewing protein gel(includes 11 constructs), ran the gel and printed the results.
We started overnight cultures of LB and Cholera and stored them in the 37 degree incubation room.
~JT,AB~
June 28, 2012
Ran the gel of the step 2 chimeric protein PCR. The bands at 2 kb were there for all proteins except A, Z9 and Z10 but were very faint. Did DNA purification of B-I. Need to redo step 2 PCR for A, Z9, Z10 and use step 2 PCR product as template for B-I to amplify yield. -KJ
Did a low melt for NucB and DNase, and cut out bands. The products are stored in the freezer. ~AB~
Did transformations of Cytr, Amylase, and DNase. This have been left in the 37 degree incubation room. ~AB, AF~
Started a 96 well plate which included LB and Cholera. Protocol from a research paper was used, and is as follows:
Froze down some colonies of V. Cholera for later use.
~AF~
June, 29 2012
We removed the 96 well plate and transformations from incubation. The transformations had no growth, and after talking to Dr. Grose, we've concluded that something in the protocol went wrong.
We redid the ligations and transformations for the 5 biofilm-disrupting proteins with Dr. Grose.
The 96 well plate of LB and Cholera showed growth, presumably biofilms, but they had been contaminated(possibly from contaminate LB), and so it needs to be done again.
We practiced with the 96 well plate, attempting to remove the LB without disturbing the biofilms. We also started an overnight of cholera to use for the second attempt. The overnights were left in the 30 degree incubator for the weekend. ~JT,AB~
July 2, 2012
We looked up each of the biofilm-disrupting proteins on the iGEM registry and found that NucB, Amylase, and deoxyribonuclease (B. sub.) are not in the registry. CytR and Dnase I were both there. -AB, KH
We set up the second attempt of the 96 well plate for the bio-film assay of cholera using cholera and LB leaving row 6 as a control. Each well was set up with the same concentration of cholera being 1:100 and then followed a protocol from a research paper (link to paper here). -JA, AF
The repeated transformations performed by Dr. Grose were all successful. We took them, and collected 8 colonies of each of the five proteins. (NucB only had 6) We then made templates for each one using 50 mics of water. We also streaked each of the same 8 colonies on new plates and incubated them so that we will have more of the exact same colonies for later experiments.
After boiling our templates, we prepared colony taq PCRs for all of the five proteins. So in total we prepared 38 samples for PCR, and ran the machine. ~JT, AB, JA (Joe Anderson)~
I re-did step 2 sowing PCR for A, Z9 and Z10 for the chimeric proteins. Also I used luxN forward and Trg reverse primer for chimeric proteins B-I for PCR using previous PCR product to amplify the product.-KJ
July 3, 2012
We ran the gels for the colony PCR's run yesterday. The images are saved on the E. colonoscopy flash-drive as Audrey1July3-Audrey4July3. Three of the colony PCR's were unsuccessful so the original PCRs were redone today. These included DnaseI, Dnase (Bacillus Subtilis), and NucB. I also started a new overnight of Cholera to use for the next 96-well plate assay on Thursday. The overnight is in the 30 degree incubator. -AB, KH
From the gels, it seems that there is a possibility that one of the NucB plasmid PCRs may have worked, the DNase I did not work, the DNase (BS) probably did not work, the Amylase and CytR did work. We started overnights using three of the different-but-one-might-have-worked NucB samples, two of the Dnase BS, two of the brightest Amylase, and two of the brightest NucB. -KH, JT (gels found in AB's lab notebook)
July 5, 2012
I ran the chimeric protein PCR's A through Z-10 from 7/2/2012 on gel and took a gel picture (see Yuri's notebook). A, Z9 and Z10 had the wrong template and there were no 2kb bands for the PCR amplification of B-I.
A gel was run with the PCR's of DnaseI, Dnase Bacillus Subtilis, and NucB. The template of NucB may have been faulty, but the other two could have worked.
July 6, 2012
I finished the biofilm assay following the protocol exactly. After reading the plate with the spectrophotometer, it seems that the protocol needs to be modified slightly. The readings should have been consistent in all wells except for column 6 which was the plain LB control but all wells were about the same. We're going to have to try again and use pipettors to carry out every step. -AB
July 9, 2012
Using plasmid preps done last week, KH set up for sequencing plasmid 3, 4, and 5 of Nuc B, plasmid 1 and 3 of Amylase, plasmid 5 and 6 of DNase Bacillus subtillus, and plamsmid 4 and 6 of CytR. We pipeted 2 sets of 2 microliters of each plasmid. One of the sets got 1 microliter of primer IG12 and the other set got 1 microliter of IG57. The black labeled tubes got IG12 and the red labeled tubes got IG57. The prepared plasmids were placed in the fridge. Notes in Kellie's notebook. -KH
Overnights were prepared of cholera for another biofilm assay. PCRs for DNase Bacillus subtilis and NucB were set up with Dr. Grose. We abandoned DnaseI because we didn't have the right template. -JT
July 10, 2012
We ran the PCR products from yesterday on a gel (Dnase Bacillus subtilis and NucB) and both were successful. The PCR products were purified and the restriction digest was run. -JT, JM
We started overnights of cholera for the next biofilm assay. They were left in the 37 degree incubator overnight. The next assay needs to be started tomorrow. -AB
We also plated cholera in plain LB in two 96-well plates to be stained tomorrow. -JT, JA
Dr. Grose discovered that other papers when doing cholera PCR do a DNA extraction procedure so we did a test PCR amplification of B and C using previous PCR's that used boiled cholera as a template.-KJ
July 11, 2012
I ran a gel of the chimeric protein PCR amplification of B and C (see Yuri's notebook). The bands were still at 1kb so tomorrow we will start from step 1 using purified cholera DNA-KJ
Hillary did low melts of NucB and DNase BS. Afterwards I performed ligations of them(JT). After incubation Justin came in and did transformations of them. He put them in the 37 degree incubation room. ~JT~
Audrey finished biofilm assays with DR. Grose. They stained them, then followed protocol by removing excess LB/crystal violet via pipet this time rather than pouring. After spectrophtometry, the results were much better. Control and biofilm wells were different readings, as expected. We should continue using the technique of pipetting out the solution as it gives better and greater consistency in our results. ~JT~
July 12, 2012
Dr. Grose and I ran step 1 of the sowing PCR for all of the chimeric protein primers. The purified cholera DNA did not work so we boiled freshly streaked cholera.-KJ,Docta Grose
The transformations failed. We redid the ligation and it is incubating. Tomorrow the transformation will be redone.
~JT~
July 13, 2012
Friday the 13th!!! All PCRs worked!!! Dr. Grose performed low melt gel on Cholera Natural system group PCRs. Joe Performing dye and washing's for Biofilm 96 well tray (incubated for 2 days to produce thicker biofilm) ~AF JT JA
Step one of the chimeric protein PCR worked for all constructs except E (see Yuri's lab notebook for picture). The products were purified and used as primers for step 2 of the sowing PCR and step 1 PCR was re-done for construct E.-KJ
July 14, 2012
The transformations were taken out of the incubator. Neither of them had any growth. The overnight of cholera was taken out of the incubator as well and the control with plain LB had growth which indicates contamination. A new overnight of cholera was started and placed in the 30 degree incubator to be used for biofilm assays on Monday. -AB
July 16, 2012
Ran sewing PCR products on gel. Gel showed multiple bands and was confusing to read but E worked for step 1. Redid the sewing PCR of B and I with Trg reverse. ~ KJ and Yuri not Gagarin
July 17, 2012
Plated 3 96 well assays of cholera and LB. They will be incubated for two nights to allow more biofilm growth.
~AB, JM~
Did a PCR product cleanup on PCR product E ~ Yuri the Conqueror
Did a PCR of NucB and DNase1, ran them out in a gel, purified the PCR products, and performed restriction digest on them.
~JT~(with Jordan)
Ran step 2 sewing PCR B and I (with 1 ul step 1 PCR product and 1ul ddH20) product on gel, no results, need to consult with Dr. Grose.-KJ
July 18, 2012
Ran the restriction digest of DNase and BS out on a low melt gel with Jordan. One of the bands was really light so it was hard to cut out but the other one was good. There was also a slight mix up about which protein was in which lane but luckily they use the same restriction enzymes so it should be okay even if they were mixed up.
JM (with Jordan)
July 19, 2012
We re-did step 2 PCR of all the chimeric proteins with Dr Grose's expert assistance.-KJ, YC, Dr. Grose
July 23, 2012
-We ran a gel for the step 2 PCR reaction done on July 19 (see Yuri's notebook). Most lanes had bands at the right length except for Z9 and Z10. We DNA purified protein constructs A-I and Yuri looked up the length for each individual construct and recorded it in his lab notebook.-KJ, YC
July 25, 2012
-I ran a restriction enzyme digest with Jordan for chimeric protein constructs A-I using SacI and XbaI and did a restriction digest of the pLAT plasmid using the regular proticol. I also ran another step 2 sowing PCR of Z9 and Z10.- KJ
I finished a biofilm assay with successful spectrophotometer readings. (Picture) -AB
We prepared competent cells for cholera transformation using protocol found by Yuri and Audrey. Dr. Grose grew the Cholera in LB earlier in the morning, and then i came in and finished the procedure with her. We added the plasmids with pLAT, then continued with normal transformation protocol to allow the DNA to recover. We plated this product on a AMP resistant LB plate and put it in the 37 degree incubator over night. ~JT~
Plated out two more 96 well plate assays. They are incubating for two days to allow biofilm growth. ~JM~
July 26, 2012
We ran the transformation of NucB and DNase BS again. We had Jordan do the work with us watching to make sure it was done right since we have had several problems. Those two plus the control are incubating in the 37. ~JM, YC~
-I ran the step 2 sowing PCR for Z9 and Z10 on the gel and it failed miserably. I also made a low-melt to run the other chimeric protein constructs but did not have time to run it today.-KJ
July 27, 2012
I ran the low-melt of protein constructs A-I and the pLAT plasmid. We took a picture (Yuri's notebook) and the bands for the protein constructs were very faint and not visible under the hand-held UV lamp. Dr. Grose took gel slices where the bands were supposed to be. There was no band for A under the powerful UV light so we didn't make a gel slice, C gel slice got lost somewhere between the dark room and the freezer in 888 and I might not be concentrated enough. All in all we have constructs B and D-I to ligate. Dr. Grose said to use 5 microliters of insert and onlt one microliter of plasmid because of the low insert concentration.-KJ
We plated a new 96-well plate with Dr. Grose, practicing sterile technique. -JT, AB, JM
July 30, 2012
Plated two more 96-well plates with cholera sitting in the 37 degree incubator. We also started overnights of cholera, both in plain LB and in LB/amp. The PCR for NucB and Dnase (Bacillus subtilis) was redone. -AB, JM, JT
We performed ligations of chimeric proteins B and D-I using 5 mics of vector and 1 mic of plasmid because of the faint bands in the gel slices. Then we performed transformations on those ligations (plating 250 microliters of cells) and are in the incubator.-KJ, YC
July 31, 2012
We checked the transformations and only E, F and I worked. Yuri and i began Colony PCR for the transformations that worked. We chose colonies, streaked them out, and made template in preparation for the PCR. ~JT, YC~
We re-did step 1 PCR for Z9 and Z10. We also did step 2 PCR for A and C.-KJ, JM
We re-did the transformations that didn't work (B, D, G and H) and we plated all 500 mls cause 250 didn't work last time.-KJ, YC
August 1, 2012
August 2, 2012
We ran the colony PCR's on gel for E, F and I and we ran the step 1 sowing PCR for Z9 and Z10. We took a picture of the gel (see Yuri's lab notebook). The colony PCR's did not work (one of the wrong primers was used) but Z9 and Z10 worked and had big, bright bands at 500kb.-KJ, JT
Only G and H had growth on the transformations (two colonies each) while the control had about 8 or 9. So we set up colony PCR for the four colonies and did a new ligation for B, D, G and H. We also set up step 2 sowing PCR's for A and C.-KJ, JT
August 3, 2012
I did PCR purification of Z9 and Z10 and started a step 2 sowing PCR reaction. I also ran the gel for the four colony PCR's and for step 2 A and C (see Yuri's notebook). We are not sure if the colony PCR's worked, we sent a picture of the gel to Dr. Grose. There was no band for A and a small band for C so I did PCR purification for C. Jordan and I also set up new transformations for B, D, G and H using the ligations made on August 2 and they are in the incubator.-KJ, JM
August 6, 2012
We pulled out the transformations from the incubator on Saturday, all had growth from 1- over 10 colonies. We are not sure what primers we should be using so we did a test colony PCR of one colony from B and D using both IG11/12 and IG12/57 to see which ones work. We also ran a gel of the step 2 PCR of Z9 and Z10 (see Yuri's lab notebook for gel picture). The gel was totally empty, the ladder ran but nothing else did.-KJ, YC
August 9, 2012
The 96 well plate assay has had enough problems that we will be abandoning that method for the moment and we will use a new method which Dr. Grose has outlined for us. This is found on a paper which i will keep in Audrey's lab notebook. -JM
August 13, 2012
Made new Lb for our assay. It contains 15 μg/mL instead of the normal 100 due to the fact that cholera can't stand the higher concentration. -KN, JM
August 15, 2012
I performed the transformation of Plat into E. coli for use in our assay. -JM
August 16, 2012
Dr. Grose and I set up the overnights for the new assay. We set up overnights for CytR 4 and 6, DNase 5 and 6, Amylase 1 and 3, Nucb 3 4 and 5, E. coli with Plat plasmid, and Cholera with Plat plasmid. -JM
August 17, 2012
All of the overnights except for Cholera worked great. Unfortunately we can't proceed without cholera so we will have to patch out the colonies on the Cholera plat to find out which ones have resistance. We don't have plates for the so i made plates with the 15 μg concentration to patch that out on. -JM
Box Assignments
1A-E Purified Plasmids- need sequencing
1F CytR lowmelt band
1G Amylase lowmelt band
1-9H purified step 1 chimeric proteins (A-I)
7-8G purified step 1 chimeric proteins Z9-Z10
1I NucB purified
2A-E Colony PCR
9A-B Bacillus templates
9C AiiA PCR products with 3 different B. subtilis templates (April 30)
9G PlatS
9H Signal
9I failed PCR w/ acclaimed template Homo sapiens
The Human DNA sample (labeled BG on the top, DNA on the side) is in the Cholera PCR box along with the Bacillus Subtillus template 11774. (as of 7/3/2012)
-KB updated 6/26/2012
Biobusting Protein Status
NucB- ligated- in sequencing (may not be correct)
DNAse I- ligation did not work- start from PCR
Amylase- ligated- in sequencing
CytR- ligated- in sequencing
B. subtilis RNAse- ligated- in sequencing, probably not correct, -start from PCR
Apple flavinoid- pulling out DNA has not been effective, not using this anymore
Dispersin B- e-mailing professor for template- not looking good
AHL lactonase- re-e-mailing professor for specific Bacillus subtilis strain
Savinase- working on obtaining template
Holin- PCRs have not worked, giving up on this part
Updated 6/11/12 by Kyle Jackson Updated 6/19/12 by Kellie Hecht Updated 7/9/12 by Kellie Hecht
