Team:Baltimore US/Notebook

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Revision as of 19:56, 19 October 2010

With a little guidance from some trained professionals we should be able to figure this stuff out... I think we are actually getting there!! - Dr Liz	File:Baltimore US logo.png 200px
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==Notebook==

Oct 14 @ Burkett Lab
Robert was testing our product from overlap against pcr conditions and possible errors.
Our last 4 gel electrophoresis of pcrs have raised some concerns about whether our new POLI
pstI site template is what it should be and if our primers are still good with it.
First thing we have done today "Blast" the primers against the original POLI template
to check for possible binding sites. There are a few around locations that could cause problems but
they are not closer than 100 bp to the single base mutation we made - thus shouldn't account
for the differences. Looking back at the recent pcrs, a temperature gradient hasn't been employed
against the new template yet, and we may need higher stringency in our new, bred template.
Robert set up pcr reaction for 5 reactions with varying annealing temperatures
to compare against the TAQ genomic POLI gene
Test 1 @ 58 degrees C, Test 2 60 degrees, Test 3 62 degrees, Test 4 64 degrees, Test 5 66 degrees
Results of our overlap had given us something of the right size, but not our product or the POLI
gene anymore. As the Overlap PCR has given no good results after seven attempts, and the seventh was
a counterfeit of the right size just to make it difficult to tell if the Overlap was successful, we
will be attempting a different approach. In order to make sure that we have something to submit, even
if not our intended product, we will be working on placing a POLI gene with the BB-prefix and suffix
in the required plasmid as well as trying a new method for the single base mutation. The new method
mimics a kit (Stratagene) designed to be used while a gene is in a plamid already. This procedure first
copies the whole plasmid on the inside and outside (with the mutation from the primer) and then uses dpnI
to remove the methylated original plamid, leaving only the intended product already in the plasmid.

Modified template vs. Genomic in same reaction:

Oct 13 @ Burkett Lab
results for POLI - PSTI:
odd...

Oct 12 @ Burkett Lab
Robert ran another pcr with 2 tests: diH20 25.5 ul, buffer 10 ul, dNTP 1 ul, DMSO 2.5, Fwd primer 2.5
Rv primer 2.5 ul, Pol I 5 ul, PFU 1 ul. The second test was the same except 10ul POLI and 20.5 ul diH20
pcr ran with an annealing temp of 58.5 degrees C.
got good results, ran PCR with POLI - PSTI.
Bernadette ran a gel on Promoter, Double Terminator and RBS digests cut and uncut.
RBS and Double Terminator looked fine. Bernadette cut out from gel - will redo Promoter.

Oct 11 @ Burkett Lab
Robert's pcr gel was blank except for the control lane - He'll rerun tomorrow
Bernadette did RBS digest w/ 15 ul DNA, XbaI 1 ul, PstI 1 ul, BSA .5, Buffer 4 @ 5 ul, diH20 27.5. Also
an uncut control with 29.5 ul diH20. Promoter digest was identical except used SpeI 1 ul and Pst I 1 ul -
Also an uncut control. Double terminator Digest the same except EcoRI 1 ul and XbaI 1 ul w/uncut control.

Oct 7 @ Burkett Lab

Bernadette & Robert
Gel results were not what was expected for amplification, but the product is good after gel purification
Robert tested PCR w/ original TAQ to get optimal conditions. Tested different gradients of DMSO/temps.
Bernadette ran gel on double term, RBS & Promoter

Oct 6 @ Burkett Lab

Robert's gel results from pcr (58 degrees C, 2.5% DMSO & 61 degrees, 2.5% DMSO) yield results that match
TAQ poli in size. Part extracted & purified. Next..Amplification through cloning into a cell and PCR

Sept 30 @ Burkett Lab

Amplify more template -Robert received large bands on gel. Will cut out and purify on Tuesday

Sept 28 @ Burkett Lab
Robert ran gels on Prefix & Suffix. Prefix did not come out well, Suffix fine.
Remixed primers, tested templates/all parts for contamination -zip
After further overlap investigation, Robert discovered that several changes were necessary
to see successful overlap results. 1st: Our template should be 5X the amount we had been using
2nd: Pfu Turbo is required as opposed to the Pfu we had been using.
3rd: We are to add the Primers After Cycle 15 during PCR

Sept 27 @ Burkett Lab
Bernadette & Ryan
Spectrometer readings on 2 samples Prefix & Suffix were as follows:
P1 Absorb = .818 Concen =40.8863 ug/ml P2 Absorb = .771 Concen. = 38.5439 ug/ml
S1 & S2 combined for larger sample Absorb = 1.221 Concen = 61.0552 ug/ml
Overlap pcr attempt #5.. Annealing 59 degrees C, Melt 95 degrees, Extension 72
Same basic results as before, checking the template quality

Sept 23 @ Burkett Lab
Bernadette's notes
Robert did PCR for Suffix with Alicia's assistance. Robert purified the Suffix & Bernadette purified
the Prefix. In freezer with iGEM label P1 & P2 for prefix and S1 & S2 for Suffix
New Pfusion 400 enzyme arrived -prepared for overlap Monday!

Sept 21 @ Burkett Lab
Bernadette & Robert present
Bernadette's notes
Overlap extension problems have taken up several labs. Repeated PCR results have not established
overlap success. Reran at different temps/times, confirmed integrity of materials, etc
After further research on the overlap process, Robert brought to our attention a critical template
volume error. In addition, primers are supposed to be added during the PCR reaction
and a different Pfusion variant should be used. We are now prepping for the next phase.

Yesterday's lab, I prepared prefix & suffix primers for PCR to replenish our supply.
5 Prefix reactions, labeled #1 thru 5: diH20 25.5 ul, buffer 10ul, dNTP 1 ul, DMSO 2.5 ul,
2.5 ul of FW primer & 2.5 ul Taq RM primer, 5 ul Taq DNA, 1 ul Pfu
5 Suffix reactions, labeled 6-10,identical to the 1st 5 reactions except the suffix primers were used
Taq FM 2.5 ul and RV 2.5 ul. I also ran a template control labeled 11 and a primer control labeled 12

Hot started PCR at 98 degrees C for 1 minute - paused, added Pfu, PCR ranging from 57-98 for 35 cycles
Today, I ran the gels for the reactions. Suffix problem (labeled 6-10) that I'll rerun Thursday.
Prefix looked good. I cut out the gel for purification, also to be completed Thursday.

Sept 9 @ Burkett Lab
Robert & Bernadette present
Bernadette's notes:
Robert ran gel from overlap with not the results expected - faded bands & not the correct size
We discovered that overlap PCR not run at optimum annealing temp of 59 degree C where we
previously had success.
We prepped 4 more experiments - 2 as controls.
Bernadette prepped #1: diH20 31 ul, buffer 10 ul, dNTPs 1 ul, Fw Primer 2.5 ul, Rv Primer 2.5 ul,
DNA Prefix 1 ul, Dna Suffix 1 ul. Hot Shot start @ 98 degrees before adding enzyme, 1 ul Pfu
Bernadette prepped for Control PCR #4, identical to #1 but no DNA & 33 ul diH20
Robert prepped #2, identical to #1, except doubled DNA to 2 ul each & 29 ul diH20
Robert also prepped Control PCR #3 with no primers & 36 ul diH20.

We are very optimistic that our PCR gels will confirm our expected overlap results Monday

Sept 7 @ Burkett Lab
Robert, Ryan & Bernadette present

Previously cut DNA from gels. Ryan & Robert performed extraction with a 'Spin Gel Extraction Kit'
Spectrometer readings confirmed we have samples!
Prefix = 1.777 absorption w/concentration 88.8718 ug/ml of sample
Suffix = 1.762 absorption w/concentration 88.0820 ug/ml of sample

PCR prepped for overlap

August 31 @ Burkett Lab
Notes from Robert Buck:
Today, we have re-run the gel on the two fragments of the modified TAQ part, and the results show the
experiment to be ready to move on. We have the modified pieces and have started running a gel to purify the
products before PCRing them together. I am uploading a picture of the gel re-run (which needed re-running
due to some bad buffer the last time.) The first two lanes are 1kb and 100 bp ladder, the third is prefixed TAQ,
and the fourth is suffixed TAQ.

August 30 @ Burkett Lab
WOOHOO!!! Once more into the fray...

It was a great meeting. The enthusiasm of fall semester starting filled the halls of the school, and created a warm environment to reconnect with the group after a month's downtime in a land of transport troubles.

Progress is continuing to be made on PoliColi. Some buffer problems have been addressed, and we're hoping to have successful results showing within the next week or so. We still need to ligate the ignition and then we'll need to put the whole thing into the C3 plasmid, and test it. We have had a NY iGEM team volunteer to test, but we'll need to test compared to commercial product, and folks within DIY-Bio have offered to test as well. We'll also need to show our purification protocols and write up our characterizations.

Some work on the wiki is needed. We still need pictures uploaded, and even taken of the participants. Please update days with your journal entries of what has occurred and your reflections in regards to troubleshooting so we can show a path to those without these experiences. The simplistic modeling flowcharts need to be constructed for the various parts as well.

More supplies were identified and acquired for the TraJ focus experiments being conducted at the Towson laboratory, where Liz, Lisa, Patrick and Duke will be toiling away in between classes.

Tom also discussed the ideas of working with the local high school teachers, forensic academy and DIY-Bio contingent to make up some of the kits, Miles has been working on in connection with some small experiments. Patrick and I have proposed an smaller dry run for the DIYFest coming up in October. We'll need input from Miles to see if this might be doable, in conjunction with the Harford Hackerspace contingent.

Ryan, Tom and Bernadette discussed the BioSafety Review Board and the questions iGEM has presented us with in this self-audit. Ryan also brought up his recent enrollment in ABSA and invitation to this years conference in Denver at the tail of September, suggesting it may be a perfect opportunity to show our level of commitment to proceeding cautiously and in good measure. He also reminded folks of the Public Presidential Summit on BioEthics in two weeks on the 13th & 14th in Philadelphia, to see if anyone might be interested in going.

We're awaiting permission from CCBC and TU public affairs officials still to find out whether we're clear to have the documentary folks from Germany here at the end of October that will be following us in our last few weeks up to the jamboree at MIT. Another intriguing development in media exposure.

We will continue to schedule Strategy Nights from 8-10 on Mondays at the CCBC lab (room d206) and will have workbench hours in the lab on Mondays from 12 - 5pm, as well as Tuesdays from 1 - 5pm, for those of you whom might be able to join us to work on your technique and get some of this out of the way. With this in mind, we need to keep our workflow in mind to make certain our projects will be at state that can be put away for a few days when we walk out on tuesday afternoon, since Tom's schedule is booked up with active classes throughout the week. We need to keep this in mind in regards to the equipment and supplies we're using as well, since the novices will be playing on off-hours.

This has been your BioMore Update on your Baltimore - US iGEM 2010 team....

Over and out...

August 23 @ Burkett Lab
Parts to be transferred to Roberts Lab:

Sketch of the TraJ xo protocol:

Donor: WTF+ Kan Recipient: OriT Amp, Chlor

1. Transform TraJ into donor
2. Conjugate F into donor
3. XO TraJ
4. Select K, A, C.

or:

1. Conjugate WTF to recipient.(A,C)^R
2. Recipient RecA- has OriT, inducible TraJ T^R, Select A,C,T.
3. Scrape colonies, pool.
4. Make competent.
5. Transform in TraJ-Kd4 PCR product, select TACK or select K, then TAC.

Exp: see transfer of A,C iff arabinose induced.

August 16, 2010 @ Burkett's Lab

Bernadette's notes:
Robert ran gels from our rxns on Aug 12th - success- Robert analyzed

We tweaked rxn conditions for optimal DNA amplification.

Bernadette's rxn primers included TAQ Rm and Bb Suufix + TAQ Reverse
PCR temps ranging from 57 degrees C to 98 for 30 cycles

Robert's rxn primers included Fwd Poll Complement and TAQ Fm
Robert ran separate PCR

August 12, 2010 @ Burkett's Lab

Bernadette prepped Control for PCR (Color Coded Red) with 34 ul H20, 2ul Control Template DNA,
10 ul 5x Pfu HF Buffer, 1 ul 10mM dNTPs, 2.5 ul Primers, .5 ul Pfu DNA Polymerase

Cycling 72 - 98 degree C, hold @ 10 degrees C

Also ran Agarose gel for PCR 2..
prepped 2 gels - one refrigerated for future use
Observed apparent DNA fragment, but will re-run samples on Monday with ladders to confirm results

August 11th, 2010 @ Burkett's Lab

Bernadette's notes:

PCR from Aug. 10th unsuccessful due to the low PCR temp of 72 degrees C. New primers received require low
temp of 69 degrees.
Prepared PCR 1: Rxn 1 (colored coded Black) included 2.5 ul Taq DNA, 10 ul Pfu buffer
25.5 ul H20, 1ul dNTP, 5 ul (diluted) Fwd Poll Complement, 5 ul (diluted) TAQ RM, 1 ul Pfu.
Rxn 2 (Color coded Blue) was identical to Rxn 1 except it included 1 ul DMSO & 24.5 ul H20
3rd group (Color coded Red) was a no DNA group
4th group (Color coded Green) was a no enzyme group

Ran a Touch Down PCR ranging from 61 - 98 degrees C

Prepped PCR 2:
Rxn 1 (Color coded Light Green): 2.5 ul Taq DNA, 25.5 ul H20, 1 ul dNTPs, 5 ul TAQ FM (diluted)
5 ul Bb suffix+TAQ RM (diluted), 10 ul PFU buffer & 1 ul PFU

No DNA rxn (Color Coded Orange) prepped w/ 28 ul H20 & obviously no TAQ DNA

No PFU rxn (Color Coded Brown) prepped w/ 29 ul H20 & no PFU

Touchdown PCR --cycles ranging from 61-98 degree C

Agarose gel ran for PCR 1 completed 8-11

August 10th, 2010 @ Burkett Lab

Robert's notes:
New dNTPs and Primers for PCR received and aliquots made of each.
PCR optimization can be started back up once again, now that uncontaminated
materials are available again. Testing the necessity of a 2:00 minute denature time
at the start of the PCR program, as well as testing three parts
(J23056, J23031 & J23008) for DNA after boiling mini-prep from a few days ago.

For the PCR, I am using concentrations of .5 uM for primers and 200 uM for dNTPs,
a 1:1000 dilution of the three parts for boiling prep DNA, and for the other
tube's DNA, a 1:10000 dilution of J04450.

Duke ran a gel for the PCR products from last night's meeting, where he and
Bernadette had started work on the TAQ project's first step. The results were
less than he had hoped, based on his reaction to the gel.

Patrick's notes:

August 9th, 2010 @ Burkett lab

Bernadette's notes:

Duke ran PCR #1

Rxn 1 consisted of 1 ul PFU, 2.5 ul Taq DNA, .5 ul FM, .5 ul RM, 10 ul PFU buffer
.5 ul dNTP & 35 ul H20.
Rxn 2 was identical to Rxn 1 except it contained 1 ul DMSO & 35 ul H20
A No DNA sample was produced and A No PFU sample was also produced

Bernadette ran PCR #2

Rxn 1 consisted of 1 ul PFU, .5 ul FM, .5 ul RM, 10 ul PFU buffer, 2.5 ul DNA, 35 ul H20
Rxn 2 was identical to 1 except that it contained 1 ul DMSO & 34 ul H20
A No DNA sample was produced (no DMSO) & a No PFU sample produced (no DMSO)

All 8 rxns ran through 30 cycles ranging from 72-98 degrees C

August 3rd, 2010 @ Burkett lab

To determine validity of Castenholz media, Bernadette performed an inoculation
of Thermos Aquaticus/Taq. Due to the non antibiotic resistant Taq,
inoculation completed under sterile conditions/hood with all equipment, material
and limbs under hood cleaned with 70% isopropanol w/autoclaved pipettes.

Taq kept at -85 degrees C, heat blocked at 37 degrees C for a minute

15 ml media was combined with Taq

Incubating overnight @ 70 degrees C to observe for Taq growth.

Patrick completed DNA extractions using 23007 and 23030
Bernadette ran a gel to determine DNA presence.
DNA successfully extracted confirmed by Electrophoresis.

July 29th, 2010 @ Roberts Lab / Towson
Dr liz is wondering if anyone is bringing materials from CCBC over to the TU lab today. It is currently 1:50pm at the time of this post. Please email Dr Liz and let her know what, if anything, you are planning for today.

July 28th, 2010 @ Roberts Lab / Towson
In Attendance: Dr Roland Roberts, Dr Liz, Dr Schiefele, Patrick, Ryan, Duke

Dr Liz introduced this group of iGEM folks to Dr Roland Roberts. Dr Roberts is willing to have a small cadre of the iGEM Team Baltimore work in his lab. Because Dr Roberts is not officially an iGEM Advisor, we are going to be especially considerate about using his lab space. We are not going to have any more people come into his lab space at this time. There may be room for one more person to work in the Roberts' Lab at a later date.

Dr Roberts has a very disctinct protocol in his lab, and he was kind enough to distribute Lab Instructions to all of us -- these are what he gives his own students when they first comoe into his lab. We are very grateful for Dr Roberts' support, and we will show that by being especially sensitive to his lab policies and procedures. Dr Roberts keeps a sterile working environment. PCR reactions and cloning are always done under the hood. His lab only uses filtered tips. Dishes do not stack up in his sink. We will help do the autoclaving. Ethidium Bromide is done in only one area of the lab - it is stained into gels after they are run, rather than put into the gel agarose. The reasons for all of Dr Roberts' protocols are completely obvious. (Even if they weren't, we would follow them anyway!)

We will have to provide ALL of our expendables - from gloves, tape, felt-tips, foil for autoclaving, tissue wipes, clips and storage bottles, to all reagents we need for reactions. That means gel agarose, molecular ladder, restriction enzymes, ligase, competent cells, antibiotics, cell culture plates and agar, DNA oligos, primers, dntp's, polymerase, BSA, buffers for PCR and Restriction digestion reactions. We also need to bring over a gel apparatus or two (he has ample power supplies), pipetmen, filter tips, tubes of all sizes, work blocks for holding tubes, chemical jars (orange lids) if we need to mix up own own solutions. Other needs will become apparent as we begin to perform experiments in the Roberts lab.

We do not have to provide our own TAE, TBA or paper towels. These items are cheap enough that Dr Roberts can let us use his. We will not bring ethidium bromide for now. Maybe later.

We may use Dr Roberts' glassware. If you break it, Dr Liz buys it. Please be careful! We have access to PCR machines, rockers, water bath, centrifuges, spinners, cold centrifuge, fridge, freezers (both -20 and -80), microwave, autoclave, and the gel imaging system. The cell culture room is on the 4th floor of Smith and is open all the time for anyone's use.

We may also use the PC's in the lab for lab-related work or checking email. (Please do not update your facebook account on Dr Roberts' PC's!). He has two pipet stands - one has been spoken for by Dr Liz. Sr Jim Saunders, Director of the MB3 Program, has gotten lengthy wish lists over the last few days from Dr Liz, and he has already provided storage boxes for tubes. He emailed Dr liz yesterday and said he has more stuff. Dr Saunders is sure to be a great resource.

In Dr Robert's lab, it goes without saying that Dr Roberts' say is final. Otherwise, Dr Liz's say is final. It is her reputation on the line at the Towson Lab space, and anything the iGEM team does will reflect directly on Dr Liz. Anyone who cannot work under that caveat should bow out now from the Towson Lab space.

On a more upbeat note, the Towson Lab will have results to report soon, we hope!

July 27th, 2010 @ Burkett Lab
Day Shift: Patrick, Robert, Duke and Ryan

Running restriction digests on ANN parts for validation. Problem with <partinfo>J23030</partinfo>: restriction digest shows only a faint band of genomic DNA in lanes 2 & 3 (cut, uncut with Spe1):

http://imgur.com/ePNEO.jpg

Will run a larger gel tomorrow with all ANN parts to confirm.

--Pon 22:40, 27 July 2010 (UTC)

Ryan ran the transformations for the PoliColi Ignition Ligation. (<partinfo>R0010</partinfo>, <partinfo>B0034</partinfo>, <partinfo>PsB1C3</partinfo>)
https://static.igem.org/mediawiki/2010/c/c2/BbPart_%28Ignition-R0010%2CB0034%2CPsB1C3%29Transformation.pdf
When we return to the lab on thursday, cell plates must be checked from incubator. Looking especially for results on RXN plate, sample should be run for electrophoresis sizing after additional culturing.

July 26th, 2010 - Burkett Lab
Attendence: Robert, Duke, Ryan, Patrick

Ryan ran the Photospectrometer analysis on the Bb DNA that's been cultured for the ANN and PolIColi ignition components. Results are as follows...

<bbpart>J01001</bbpart> A260 = 1.03 Concentration= 51.57 ug/ml Concentration = 0.05 ug/ul
<bbpart>J01003</bbpart> A260 = 2.37 Concentration = 118.55 ug/ml Concentration = 0.12 ug/ul
<bbpart>J23007</bbpart> A260 = 1.33 Concentration = 66.72 ug/ml Concentration = 0.07 ug/ul
<bbpart>J23008</bbpart> A260 = 2.52 Concentration = 125.74 ug/ml Concentration = 0.13 ug/ul
<bbpart>J23022</bbpart> A260 = 1.87 Concentration = 93.46 ug/ml Concentration= 0.09 ug/ul
<bbpart>J23030</bbpart> A260 = 3.11 Concentration = 155.94 ug/ml Concentration = 0.16 ug/ul
<bbpart>J23031</bbpart> A260 = 0.14 Concentration = 7.11 ug/ml Concentration = 0.01 ug/ul
<bbpart>J23032</bbpart> A260 = 2.01 Concentration = 100.36 ug/ml Concentration = 0.10 ug/ul
<bbpart>J23036</bbpart> A260 = 2.08 Concentration = 104.07 ug/ml Concentration = 0.10 ug/ul

<bbpart>R0010</bbpart> A260 = 1.44 Concentration = 72.19 ug/ml Concentration = 0.07 ug/ul
<bbpart>B0034</bbpart> A260 = 2.55 Concentration = 127.36 ug/ml Concentration = 0.13 ug/ul
<bbpart>B0015</bbpart> A260 = 2.43 Concentration = 121.23 ug/ml Concentration = 0.12 ug/ul

July 23rd, 2010 at Towson Campus
In Attendance: Liz, Roland Roberts, Jim Saunders

Dr Liz met with Dr Roberts and was given the tour of the lab space and equipment available for Team Baltimore to use if a subgroup wants to work at TU. All the usual equipment is available except Dr Roberts want Team Baltimore at TU to bring their own set of pipetment, and of course, supply its own needs for expendable items. Dr Jim Saunders has taken a copy of Team Baltimore at TU's wish list, and says he can provide a set of pipetment, and tips/tubes without much trouble. We also need to take a gel electrophoresis apparatus, because Dr Roberts has a limited supply and other students use them frequently. He does have an extremely large one he said we could use.
Dr Roberts wants to meet with the students who wish to have access to his lab, sooner rather than later. It is important that Team Baltimore bring reagents over to the Roberts Lab and begin to use some of the space Dr Roberts has set aside for us, in order to demonstrate that we are serious about using his lab. Anyone who would like to work at the TU site sometimes should contact Dr Liz immediately.
There is a DNA sequencer and a high-throughput DNA seperator that is accurate to 1 bp for oligos between 100-650 bp long. If Team Baltimore wants to use this machine, that is possible. In that case Dr liz will talk to her Dean at TU to acquire funds to help share the cost of using it.

July 20th, 2010
In Attendance: Gary, Bernadette, Tom, Patrick, Ryan
During day shift, Robert finished working on PCR optimizations. While Patrick and Ryan, troubleshot their primers and put the order in so that they can begin PCR reactions when they return on monday.
Bernadette concentrated on her Ligation reactions for the PolIColi project.
Ligation reaction (COLOR CODED RED)included: 6 ul R0010 promoter, 6 ul B0034 RBS,
2 ul PsB1C3 chloremphenacol resistant plasmid,2 ul buffer, 3 ul H20 & 1 ul ligase enzyme
For comparison, I also completed a 'No Insert' group (COLOR CODED TEAL/BLUE)using 2 ul plasmid, 2 ul buffer,
15 ul H20, 1 ul ligase
In addition a 'No Enzyme' group (COLOR CODED DARK GREEN)was completed that included 6 ul R0010 promoter,
6 ul B0034 RBS, 2 ul plasmid, 2 ul buffer, 4 ul H20
The 3 groups were incubated at 16 degrees C overnight

Gary got caught up on project status and development.

July 19th, 2010
In attendance: Miles, Patrick, Robert, Duke, Liz, Tom, Bernadette, Steven
Steve transformed <partinfo>J23030</partinfo> as it was the only DNA from last week's transformations that did not develop any colonies.
Bernadette began restriction digestions for the Bbparts for the PoliColi project.
Part #'s
<partinfo>R0010</partinfo> The LacI Promoter
<partinfo>B0034</partinfo> The Strong RBS
<partinfo>PsB1C3</partinfo> A Chloremphenacol Resistant Plasmid Backbone
<partinfo>B0015</partinfo> The Double Terminator
<partinfo>R0010</partinfo> and <partinfo>B0034</partinfo> are meant to be ligate into the <partinfo>PsB1C3</partinfo> as all the initial PoliColi Bbparts had a native Amp resistance and one had Amp and Kanamyacin.
The <partinfo>B0014</partinfo> will be ligated with the PoliColi NewPart once the Overlap Extension Point Mutation PCR is completed.

Restriction digestion included the following: R0010 Promoter (COLOR CODED BLUE): 5 ul DNA, 5 ul buffer#4,
37.5 ul H20,cuts made with 1 ul EcoRI and 1 ul SpeI enzymes, and .5 ul BSA The result will be the insert
In a separate reaction B0034 RBS (COLOR CODED PURPLE): 5 ul DNA, 5 ul buffer #4, 37.5 ul H20, 1 ul XbaI,
1 ul PstI,.5 ul BSA
The 3rd reaction PsB1C3 plasmid(COLOR CODED LIGHT GREEN): 5 ul DNA, 38 ul H20, 5 ul buffer #4, 1 ul EcoRI,
1 ul PstI, no BSA
Incubated overnight 37 degrees C

Ryan and Patrick began working on Primer design for their various PCR projects.
Miles showed off initial development of Gel Electrophoresis Kit and Power Supply. Discussed PCR construction with Tom, as well as introduction to Oligonucleotide synthesis.

July 12th - 15th, 2010
Dayshift Lab Techs began DNA Preparation of various identified BbParts. Robert and Duke finished with Boiling Prep.
Evening Lab intermittently closed with Tom unavailable for lab supervision.

July 8th, 2010
In Attendance: Gary, Patrick, Robert, Lisa, Liz, Steven Strategy Meeting in spare room. Patrick outlined strategy of ANN (Artificial Neural Network) based on Berkeley's previous work. Discussed use of conjugation plasmids for weight assessment.

July 6th, 2010
Can't quite remember back far enough to state what occurred. Got to remind everyone to update the notebook wiki page as during the evening's in question.

July 1st, 2010
Ryan didn't make it, but I hear something happened.
June 29th, 2010
In attendance: Day - Duke, Tom, Patrick, Ryan, Robert
Evening: Gary, Liz, Tom, Patrick, Duke, Ryan

During the day-shift Robert continued to work on the PCR optimization, with the second set of Gels showing a continued contamination of one of the main components, pointing possibly to the template mixing into the dNTP's. Patrick helped Tom with some Thermophilic cultures, and Ryan completed the Plasmid Transformations of T3, K3, & C3 along with the Batch prep of the CAT8 plasmids.

We caught tonight's attendees up to speed as to where the group stands on it's individual projects. Then together we discussed in detail how to break up the foundational work-flow and what steps need to be accomplished to make them happen. Patrick detailed some of the foundational work previously performed by the earlier iGEM teams on Artificial Neural Networks.
DIY-Gem notes: Creation of Pol-1 Taq Polmerase in Bb format J04637.1
1. Amplify Pol-1 gene from T. Aquaticus

  Convert to Bb format - Add Bb Prefix and Suffix 

  Change internal Pst-1 site - Single BP Mutation 

2. Characterize Part

  Error Rate? 

  Activity 

  Etc... (Versus Lab Ordered Purity) 

3. Educational Supplementation

  Techniques - How to do a single point mutation 

  Tools Discussion / Software & Hardware 

DNA Computing Notes: Turing Automatons w. Back Propagation
1. What Species is it?

  Identifying parts available. 

  Identify parts to be made. 

  Assemble parts. 

  Build Assemblies. 

  Test & Characterize System. 

What is a Neural Network? The XOR switching system.

 - Addressable Conjugation. 

 - RNA Hairpins. 

 - HSL Senders / Receivers. 

 - Others. 

 - Attaching varied weights among various pathways. 

 - Summing. 

 - Thresholdiing. 

 - Back Propagation Control through Lysing (Cellular Suicide model). 

June 28th, 2010
In attendance: Day - Duke, Robert, Patrick, Robert, Ryan, Tom
Evening: Miles, Robert, Patrick, Tom, Ryan, Burnadette

We welcomed a new comer Bernadette Gallagher. And spoke more in depth about project design and workflow. Tom helped get our new team mate oriented, while Miles, Patrick and Robert worked on flushing out conceptual foundations for their projects of interest.

Ryan - Performed Restrictions (e/p)and Ligations on Pb1t3,c3,k3 while assisting the batch prep upon the CAT8 plasmids, performed first step lysis to instruction 14 putting DNA in alcohol.
June 24th, 2010
In Attendance: Patrick, Robert, Tom, Duke, Ryan

After a roughly a week's break from official activities the team met to discuss strategies and tactics.
A few hands have been taking up the lab tech training from Tom and Duke throughout the lazy summer days, while doing so they revisited the basic procedural steps of ligation, gel-phoresis, transformation, as well as the tasks of mixing and pouring media, basic chemical preparation, as well as safety and hygeine in the lab. Tom has also been introducing us to the concepts behind workflow and how to divide and stage the various processes towards a greater project concept.

Miles suggested that he will be working towards several ideas in hardware project construction from a PCR unit to a DNA synthesizer. We discussed in depth Patrick's idea for the creation of a cellular automaton. He broke down the previous tactics in DNA computing where everything is synthesized then re-sequenced for the answer, then posited the creation of an Artificial Neural Network type approach in utilizing various signaling proteins to reach consensus and establishing some way to create back propagation of error.

We've decided to restrict lab nights to Monday and Tuesday from 7-10pm, with a floater strategy meeting on Thursday nights so we can stimulate thought with a change of environment. With that in mind we are back tonight and look forward to seeing those of you who can make it in to keep cranking on our technique.

June 16th, 2010
In attendance: Lisa, Ryan, Tom, Colin (for a short time), Duke (for a moment)
Missing: Steve

Earlier in the day, Robert and Ryan performed a mini-prep of the pcr'd plasmid backbones 3A1(2) and 5A1(2). Robert also began working on finding optimized conditions for pcr.

With the small turnout, Ryan began the digestion restriction with Colin's assistance. We combined the 2 strains of 3A1 and 5A1 together, then put together 3 - .8 eppendorf's with 50 ul of rxn ingrediates.

(2 tubes of positive control of both along with 1 negative control that contained no enzymes and 40 ul of H2O)

5 ul of DNA
1 ul of Pst
1 ul of EcoR1
5 ul of Buffer
38 ul of H20

50 ul - Negative Controls Labelled with minus sign.

After preparation, the cells were put into the pcr block for a 30 minute heat cycle.
Tom will be heading to Arizona, tomorrow, so we called it a night so he could pack and go.
It has been suggested that we run a "Low-Heat Agar Gel" so we can find and physically splice out the appropriate sized plasmid structures.

June 15th, 2010
In attendance: Patrick, Ryan, Duke, Tom, Steve
Missing: Colin, Liz, Scott, Andy

Tom says results seemed to have worked for all of the transformations this time. Some better then others.

Steve asks the question.. "Why did transformation work better this time?"
Tom discusses differences in attempts.

Plasmid contains part a, another contains part b, another part c is simply a linear piece of DNA representing a Tet resistant backbone with the EcoRi and PstI restriction sites.

We cut Part A with EcoR1 and Xba1.
We cut Part B with Spe1 and Pst1.
We cut the Linear Backbone Part C wit EcoR1 and Pst1.

Then we combined everything into one tube using 2 ul of each part into 1 tube, and added t4 DNA ligase, hoping to get a Part A joined to Part B, because the Spe1 combines with the Xba1. And the Part A would join Part C with the EcoR1 and the Part B with Part C at the Pst1 site.

(See photo of board - )

Last week, we got nothing, so why?
Where could things go wrong?
They could go wrong at the restriction digestion reaction (less likely as process is fairly robust, and had been tested the day previously.)

Ligation reaction may not have worked.. (again less likely, process is fairly robust). If the buffer isn't mixed well it may separate.
How do we test? we could perform pcr to see if the ligation took place, using primers to amplify the ligated segment checking with gel electrophoreis to identify.

Transformation reaction - (Except we ran positive controls successfully)
Still possible to have mixed up the media, since positive used Amp instead..
Tom checked the media and found that wasn't the case.
Amount of Plasmid may not have been accurate. (The only thing we didn't physically check ahead of time.)

Pure operator/Pipetting action - such as the 30 second heating followed by kill cycle.

Detection limits for Ethidium Bromide 10-15 ngs of a product.
(3600 bp ) (650 gm/b.p molar weight)
2,340,000 Grams per mole of 3600 bp molecule.
50 nanograms
(50 x 10-9) / 2,340,000 = 2.1 x 10-14 moles

Transformation rxn is 1 x 10 (-8/-9), still fairly efficient.

We did 2 things last night, we did the transformations into the stocks..

By using assembly strategies that don't use the same resistance as the parts, we can more efficiently screen out non-transformed pieces.

Previously in Genetic Engineering all parts and pieces had variorus resistances where as bb tries to create a standard.

Cells can be lost in replication due to metabolic costs, and new generations lacking the Costly programming tend to outgrow the special cells.

When designing the assembly strategy try to use a plasmid backbone with a different resistance to allow the filtration/screening of parts that didn't transform correctly.

Gel-Electrophoresis Prep Review for tonights run.

Agarose 2 % for smaller parts will create a more viscous environment.
0.8% for the larger parts will allow longer molecules to move better.

50ml final volume

Weigh out the agarose (.4 gms for the 0.8%) (1 gm for the 2%)

Emphasize again the importance of NCBI databases... http://www.ncbi.nlm.nih.gov/
With links to BLAST, PubMed, and other official literature. Program called entree which links all the information together.

When reviewing the tubes from last evening left in PCR there was difficulty in identifying the tubes that were done by each individual. We spent a little time using the process of elimination. Knowing Patrick, Steve, Miles, Ryans and Gary's without issue, we had to guesstimate which was Roberts, and which was David's. The reason we needed differentiation was to create a loading template, in one of the two different agarose preparations.

June 14, 2010
In Attendance: Tom, Duke, Patrick, Robert, Steve, Gary, David, Ryan, Miles

3 Separate transformations attempted: <partinfo>pSB1C3</partinfo> [1-3A] (ChlorAmphenacol resistant), <partinfo>pSB1K3</partinfo> [1-5A](Kanamicin resistant), <partinfo>pSB1T3</partinfo> [1-7A] (Tetracycline resistant) Control Group: PET-17B (still had separate resistance for Ampicillan) Tom will be culturing some adjacently resistant control groups for our next trial, thereby eliminating the extra variable in our testing.

Procedural experience:
Transformations using 1-3A 50uL EXPERIMENTAL
200uL EXPERIMENTAL
250uL NEGATIVE CONTROL
Cells were plated and put in the 37C incubator. Plates labeled in RED.

9 Parts for PCR - 7 Distributed amongst us. Various participants were asked to look up their function and various bp lengths.

Patrick - <partinfo>Bba_R0010</partinfo> - 200 bp
Gary - <partinfo>Bba_R0063</partinfo> - 151 bp
Robert - <partinfo>Bba_J04450</partinfo> - 1,069 bp
Dave - <partinfo>Bba_R0062</partinfo> - 55 bp
Steve - <partinfo>Bba_J23009</partinfo> - 97 bp
Miles - <partinfo>Bba_I731014</partinfo> 1,938 bp
Ryan - <partinfo>Bba_I13507</partinfo> - Intermediate Screening Plasmid - 861 bp

PCR for part [http://partsregistry.org/wiki/index.php/Part:BBa_R0063 R0063]:
Vial labeled with RED X is the EXPERIMENTAL REACTION
Vial labeled with RED Y is the TEMPLATE CONTROL
Vial labeled with RED Z is the PRIMER CONTROL

PCR Polymerase Chain Reactions

Requirements
1. Template
2. 2 Primers (limiting)
3. dNTP's (limiting)
4. Polymerase (limiting by heatcycle) Using Taq which tends to transcribe more errors.
5. Buffer (mg++) (limiting)

Primers are 18-30 bp long. Have to be at least 15 bp long. Primers bind to template DNA, one primer for each (5'/3' strands). Review: What is a base..? Sugar with A, C, T or G is a nucleotide. BP are the matching set of 5' and 3' hydrogen bonded nucleotides.

Polymerase links free dNTP's to the opened strand edges of basepairs.
Denaturation 95 - 98 degrees c... DNA Melts
Anealing 40 - 65 degrees c... Primer Binds
Extension 72 degrees c... Strands Extend
30 second cycles...

As the DNA heats it denatures then as it cools the primers anneal and polymerization takes place creating twice as many strands. The strands are held together only by hydrogen bonds, making it very easy to melt and reform. The polymerase acts like a little machine stitching the free dNTP's into the template.

BioBrick plasmids have bb prefix and suffix. Primer binding sites vf2, vr. Theoretically any area can become a primer site.

With PCR we can identify and amplify particular strands/compositions. You have to know how much of the requirements to use. Gary asked if Tom could show us where to find the optimized volume information at, and he agreed to pull out the research to show how to optimize.

With PCR you can also design primers and introduce mutations. If primers are too short they can bind to the wrong spot. Actual reactions may vary based on bp length and combination. Usually they are tweaked for the optimal over a series of trials.

PCR Reactions (Specific to this evening's trial)

34mM of dried DNA in primers ($12.50)

Several companies provide these primers: invitrogen is an example.

34 x 10-9 moles / 100 x 10-6 liters = 3.4 x 10 -7 moles/liter

Tomorrow we will look at the parts we pcr'd and then see how the bp length matches. If nothing happens its probably a pipetting error. If too many bands it may be an annealing issue.

We will also look at the cells we plated of the various transformed parts, 3A, 5A, 7A, and see again if any of the colonies grew.

June 10, 2010
In attendance: Duke, Gary, Robert, Patrick, Ryan, Miles, Steven, Liz, and Colin.

Tonight we tested the hypothesis that the plasmid did not code for the antibiotic resistance/that we did not use enough DNA. A Restriction Digest reaction was set up on the ligation reactions completed on June 7. The reactions were cut with EcoR1 and Pst1. The reactions were allowed to incubate at 37 degrees C for one and a half hours. The digests were then ran on a electrophoresis gel. No DNA was present as there was not enough DNA, thus proving Duke's hypothesis.

June 9, 2010
In attendance: Duke, Tom, Ryan, Patrick, Lisa, Steven. Missing: Kyle and Friend(Forgot his name)

1) Registration for iGEM site & Team affiliation, required to update team wiki.

2) Access and update wiki.. Consider stylization, as well as information content. Who is our audience? Team info - blurb and caption of team members. Couple of sentences about you and your interests. Lab Notebook? - Openwetware connection.

3) Research biobrick parts by accessing in Registry of Standard Parts. XF to see if new combination are already on file? If not begin documentation of the 3 new assemblies, with image using standard BioBrick icons. Document bp length of new parts to compare with Gel-Electrophoresis. How do we test efficacy? (Part for PPM still in brick.)

Is the media okay, is the antibiotic right. Positive tells you whether the cells themselves were capable of taking up our DNA. (Ideally, they should use the same antibiotic resistance, as our parts.

Negative groups tells us whether the antibiotic and media was effective and that the cells were not resistant to the media.

We know that cells were competent they were able to be transformed, and the antibiotic was effective. So the remaining question is whether they were able to be transformed.

The ligation's are in question. Generally the restrictions and ligation's go smoothly.

Tom's Hypothesis is questioning whether the media plates may have been mis-labeled with the wrong antibiotic resistance. Since we used a different resistance for controls, there was no telling.

Possible denaturing of enzymatic proteins from the heat shock on the initial restriction? 80c kill cycle. Vector ? - Linear plasmids - was the amount to small...? Parts - size already was confirmed, according to Tom

No Transformations.... so now what? Primers still on the way. Don't need to go to scratch.

The Core enzymes needed to perform the biobricks constructions... Spe1 pst1 ecor1 xba1 t4dna ligase polymerase

Project idea:
Polymerase in e.coli, tag, perhaps other enzymes could be self-manufactured/purified.

Basic tools/measurements

• Micropipetters [Measuring volume]
  
• Measuring Mass
  
• DV/HD camera - you tube videos and components.
  
• Mini-preps. Growing the cells and isolating DNA. Cell competency Preparations.
  

Lac polymerase - DIY-GEM

How do you purify?

What do you do with it... what is the process to create a project?

Hardware only takes you so far, these 5 basic tools. Restrictions Ligations Transformations/Plating PCR Gel Electrophoresis

The core will be good technique with these processes and then the ability to understand the existing database navigation with the proper questions to yield an experiment of interest.

Research educational tools from the MIT educator on IGEM site. Should we have an e-mail/Comments section added to wiki - openwetware, so observers can ask us questions?

June 8, 2010

In attendence: Colin, Patrick, Liz, Ryan, Duke and Tom. Missing: Andy, Scott.

So tonight we came in and took the 4 ligation's/new part combinations from yesterday and transformed them into the competent cells, using a heat-shock transformation, while also preparing the Control groups (one with nothing/one with the ampicillin resistance). 3 of the parts were Tetracycline resistant, and 1 was Chlorephenecol. After the 90 minute transformation cycle we plated 6 versions of each of the 3 parts along with 1 plate of control and one negative group.

The 6 plates were done in a 0, -1, -2 dilutions in 2 concentrations one of 50 ul, and one of 200 ul.

They will be left for tomorrows team to run gel electrophoresis to determine whether they have the appropriate combination links.

June 7, 2010

In attendance: Colin, Patrick, Robert, Ryan, David, Gary, Miles, Duke and Tom. Missing: Melissa.

Tom greeted us with 4 separate sheets that contained 3 reactions each for us to begin restrictions and began heating to let the enzymes cut. A lesson in what not to do, was offered as we began the heating cycle of restriction in the PCR blocks and he timing had been set to 35 seconds instead of 35 minutes, after which it heated to a kill cycle of 80 degrees and we had to reapply the enzymes, in case the enzymes had been denatured.

We had 6 individuals building the 4 sheets, 2 in redundancy.

The legend for the various parts is as follows...

Patrick cut parts <bbpart>BBa_r0063</bbpart> and <bbpart>BBa_p0412</bbpart>, and the plasmid backbone <bbpart>pSBIT3</bbpart>.
Robert cut parts <bbpart>BBa_R0062</bbpart> and <bbpart>BBa_I13507</bbpart>, and the plasmid backbone <bbpart>pSBIT3</bbpart>.
Collin/Miles cut parts <bbpart>BBa_F2620</bbpart>, <bbpart>BBa_I13507</bbpart> and the plasmid backbone <bbpart>pSBIC3</bbpart> (labeled w/green marker).
The next step is to ligate them.

After which we had a round table discussion about what kind of projects we may follow up with and the process of using the NCBI databases to discover pre-existing sequence information related to our various ideas. One idea we have discussed was the option of creating a smoother introductory curve for fellow DIY-Bio commmunity members and the creation of home-brewed enzymes that might be to pricey for the amateur scientists. We ended the night with the beginning of the various ligation reactions, as seen above.

• Ligation Reaction for R10+462+IT3 in 'LIGATE 05X
• Ligation Reaction for A/R10+B/462+C/IT3 in 'L/LIG
• Ligation Reaction for F2620+I13507+IC3 with "Green Asetrisk"
• Ligation Reaction for R0062+I13507 in "Squiggly Sigil"

biological computation

conjugation notes