Team:The Agency Escondido/notebook

April 9

Andrew Buss prepared 120 mL of LB broth

April 10

Andrew Buss prepared two LB plates without antibiotics, using our LB plate protocol, rev 72.

Andrew Segina and Vansh Singh inoculated two ampicillin/kanamycin LB plates with E. coli containing Parts A and B from the 3A assembly kit

Andrew Buss inoculated an SOB plate with NEB10 competent E. coli

April 11

Andrew Segina inoculated an SOB plate with NEB10 competent E. coli

The plates previously inoculated with E. coli with parts A and B do not show visible growth

Andrew Segina prepared 1 L of YPD broth without dextrose

April 12

Andrew Buss inspected plates at 8:15 AM. Both NEB10 plates showed visible growth. Neither plate with Part A or B displayed growth.

Andrew Buss prepared four YPD plates from the 1L stock and mixed 4 grams of table sugar before pouring

Andrew Buss inoculated two LB tubes each with E. coli with parts A and B directly from the agar stabs included in the 3A assembly kit. The intention was to diagnose the lack of growth on the A and B plates over two days.

Andrew Buss inspected plates again around 1:00 PM. NEB10 plates showed additional growth, and the plate with Part A contained visible colonies. The plate with Part B did not show growth, however.

Grant Hassinger mixed 100 mL of LB broth and began an autoclave cycle with the media

Vansh Singh inoculated a tube of ampicillin/kanamycin LB broth from the 3A assembly kit with a colony from the Part A plate

Vansh Singh inspected Part B plate at 10:20pm. Part B plate showed visible growth.

Vansh Singh inoculated a tube of ampicillin/kanamycin LB broth from the 3A assembly kit with a colony from the Part B plate

Vansh Singh prepared a tube of ampicillin/kanamycin LB broth from the 3A assembly kit without inoculation. The tube was placed into the refrigerator

Andrew Buss inoculated a tube of LB broth with a colony from the NEB10 plate from April 10

April 13 labeled media

April 13 available media

April 16

Our refrigerated centrifuge arrived, removing the final obstacle to 3A assembly. However, the centrifuge did not consistently power up. This issue was initially ascribed to a faulty fuse.

Vansh Singh performed DNA Miniprep for self grown colonies as per the iGEM 3A Assembly Kit

April 17

Upon closer inspection of the centrifuge, it became evident that it was in fact missing a fuse. When a 20A fuse was installed, the centrifuge powered up normally. Several test runs indicated temperature control in the range of -10 to 30 degrees C, and an effective maximum centrifuge speed of 2500 RPM (despite a dial that can be turned to 6,000 RPM).

Vansh Singh transferred DNA of Part A, Part B, and NEB10 colonies to Ependorfs to find concentration of DNA

Andrew Buss prepared 100 mL of LB broth using revision 14 of our LB liquid protocol. However, autoclaving was deferred until a larger batch of material needed autoclaving.

Andrew Buss inoculated two tubes of LB broth with E. coli containing Part A and Part B from the 4/10 plates to provide additional miniprep material for the following day. Due to the scarcity of ampicillin/kanamycin media (antibiotics to be ordered soon), these were cultured without antibiotics.

Evan Santos and Grant Hassinger streaked 3 agar plates with 3 different yeast samples for colonial development.

April 18

The tubes containing Part A and Part B had not displayed visible growth from the night before. However, the tubes inoculated on 4/12 displayed growth.

Andrew Buss extracted plasmid DNA containing Part A and Part B from the cells in the 4/12 tubes using the 3A assembly kit miniprep protocol. RNAse 1 was accidentally omitted from the P1 buffer, so it will need to be introduced before transformation. The two resulting samples were placed in the freezer, labeled "ATB A" and "ATB B". Halfway through the process, the non-refrigerated microcentrifuge was relocated to the interior of our refrigerator, because its operation generates enough heat to risk damaging the DNA samples.

Before restriction can proceed with any of our DNA fragments, the concentration of DNA in the samples must be measured. Our lab does not own a nanodrop machine, so Andrew Segina will use one at a local university to perform the measurement.

When the centrifuge was moved, it did not power up. After the purchase of a replacement fuse, the centrifuge's inconsistent operation was revealed as a consequence of an internally loose power cable. Depending on the orientation of the cable, the centrifuge may not turn on.

Vansh Singh researched GFP and RFP genes for project.

Andrew Buss inoculated three LB tubes and one LB plate with NEB10 cells from the 4/10 plate in preparation for transformation the following day.

April 19

All three NEB10 tubes from 4/18 showed significant growth. The plate from 4/18 showed faint streaks but no isolated colonies.

Quantification of the miniprepped DNA samples from the 18th has been postponed to Monday 4/22. Andrew Buss and Vansh Singh chose to continue with the 3A protocol in the interim, beginning with the restriction of the supplied DNA samples.

Andrew Buss completed restriction digest of all four samples of DNA from the 3A assembly kit: Part A, Part B, pSB1C3 plasmid backbone, and RFP control. However, 22.5 uL of distilled water was pipetted into the RFP restriction reaction, as in the other reactions, rather than the 17.5 uL specifically prescribed for the RFP tube.

Vansh Singh completed Restriction Digest of DNA from kit as per iGEM 3A Assembly kit

Andrew Buss completed ligation of the digests of Parts A and B, plus the plasmid backbone, from the earlier restriction.

April 20

Andrew Buss had planned to perform transformation. The link to the competent cell production protocol was broken, so it was incorrectly assumed that the transformation protocol had been revised to include the competent cell production. The protocol was discovered in the printed copies distributed with the 3A assembly kit, and required large centrifuge tubes and CCMB80 buffer, which our lab lacks. These are to be ordered soon, but in order to expedite transformation, a New England Biolabs order for competent cells is also to be placed.

The 4/18 NEB10 plate showed uniform growth along the streaks, rather than increasingly isolated colonies along the streak. Andrew Buss recalls that he did not flip the inoculating loop after performing the initial inoculation but before streaking, an error that was responsible for the unusual growth.

Andrew Buss inoculated a second LB plate with a section of NEB10 cells from the 4/18 NEB10 plate.

April 20 labeled media

April 20 available media

April 23

Andrew Buss's LB broth from April 17 was not autoclaved because there were no other autoclave operations with which to batch it. The broth was contaminated in the intervening time and is not usable. In the future, media will be autoclaved immediately after mixing.

Vansh Singh performed DNA Ligation on the linearized plasmid Backbone of pSBCI3, and DNA parts of Part A and Part B according to revision 18 of Ligation.

Grant Hassinger reinoculated one LB tube with E. coli with parts B from Andrew Buss's

Vansh Singh and Andrew Buss accompanied Andrew Segina to a local university to use a nanodrop machine to quantify the DNA samples from 4/18. The readings on all samples were outside of the range of the machine - either our samples were too concentrated or too dilute, or impurities were introduced. Supplies for agarose gel runs are on order - we will verify that we have any DNA fragments of the correct length when the supplies arrive.

April 24

Our competent cells arrived from NEB. While we originally planned to begin transformation today, continued difficulties with the centrifuge occupied our efforts. One of six tubes was retained in dry ice inside a styrofoam ice box inside the -20 C freezer for transformation the next day - the other five were taken to a local university for storage at -80 C.

Andrew Buss prepared LB broth according to revision 14 of our LB liquid protocol.

Vansh Singh, prepared 40 uL LB broth according to revision 32 of LB liquid protocol.

April 25

Ben Jones prepared 200 mL LB broth according to revision 32 of our LB liquid protocol.

Evan Santos prepared 300 mL LB broth according to revision 32 of LB liquid protocol.

Vansh Singh and Evan Santos researched promoters with different inducers for our project. Unfortunately, our search was unsuccessful because every inducible promoter we found was induced by arabinose. We will continue this search.

Andrew Buss began transformation. Three 2.0 mL tubes were labeled and filled with the correct quantity of DNA. They were placed in the freezer when the protocol was aborted for lack of time. Tomorrow, we intend to complete transformation for the first time.

April 26

Ben Jones inoculated a new tube of LB from Andrew Buss's Part A from 4/17

Andrew Buss completed transformation using the ligated products from 4/19. 50uL remaining from each sample has been replaced into the refrigerator.

Later, Vansh Singh started transformation using the ligated products from 4/23 only to find Andrew Buss had used all but 50uL of the competent cells. Therefore Vansh Singh only completed transformation on the Transformation Control part. 50uL remaining from the Transformation Control part has been placed in the refrigerator.The LB agar plates with Transformation Control began incubation in 37 degrees Celsius at 7:30pm.

April 27

At 10:30 AM, Andrew Buss's Transformation Control plate displayed notably red colonies. However, while the Ligation Control plate displayed colonies, they did not appear red. The New Part plate did not appear to have been inoculated correctly as no colonies were evident. Vansh Singh's Transformation Control plate from the previous day included colonies, but they were not red.

At 1:15 PM, the colonies on Andrew Buss's Ligation Control plate appeared noticeably red. The New Part plate still displayed no colonies. Andrew Buss reinoculated the plate using the remaining 50 uL of the New Part cell sample and spread the cells with glass beads.

At 1:20 PM, Vansh Singh's Transformation control plate appeared noticeably red. He then inoculated a 5mL tube of Evan Santos's extra LB liquid from a red colony from the Transformation control plate.

Vansh Singh produced another batch of LB liquid. His two previous tubes of E.Coli had been contaminated because the caps of the tubes came off in the autoclave.

Transformation Results
Vansh Singh Transformation Control
Andrew Buss Ligation Control
Andrew Buss Transformation Conrol

At 2:00 PM, closer examination revealed about 5 to 10 colonies sparsely scattered about the New Part plate that did not yet appear red. Their size suggested that they had been present before the second inoculation attempt. Therefore, the planned repetition of restriction and ligation operations was deferred until the colonies grow in size and number.

Andrew Buss inoculated three tubes of LB broth (without antibiotics) with colonies from his New Part, Ligation Control, and Transformation Control plates.

Andrew Buss performed restriction using Vansh Singh's miniprep products from 4/17, because the prepared DNA supplied in the kit for Part A, Part B, and the plasmid backbone, had been exhausted in the previous restriction step. According to Andrew Segina, the supplied NEB10 cells contained the plasmid backbone, so, while unfortunately not mentioned in Vansh's 4/17 log entry, they were run through miniprep process along with cells containing Parts A and B.

As mentioned on 4/23, the concentration of these DNA samples could not be measured using a nanodrop machine, nor was there an opportunity to return to test more dilute samples. From the recorded miniprep yields of other teams, an extremely rough estimate of 75 ng/uL was assumed for later calculations: 6.66 uL of each DNA sample and 35.84 uL of distilled water per tube. No other reagent volumes were modified. No RFP control sample was used - the transformed ligation control plate demonstrated a successful ligation process.

The lab closed at 6:00 PM before the thermocycler completed the program for restriction digest. The thermocycler program will hold the digested samples at 4 degrees C over the weekend until work is resumed on Monday. At the time of departure, the colonies on Andrew Buss's New Part plate did not appear distinctly red.

April 29

At 8:50 AM, Andrew Buss's New Part plate showed a pink lawn of bacteria. This indicates that the restriction, ligation, and transformation processes succeeded, albeit at low efficiency. Pictures forthcoming.

Andrew Buss's LB tubes from 4/27 containing New Part and Ligation Control were of a pinkish hue, and bacteria that had adhered at the waterline to the inside surfaces of the tubes were strongly pink. The Transformation Control tube held clear LB and upon closer inspection there was no sign of the pipette tip that was cut for inoculation, so the media was not exposed to any bacteria.

Andrew Buss ligated Parts A and B, plus the plasmid backbone digests, from the previous day. Transformation should occur tomorrow.

Andrew Buss worked to improvise an agarose gel setup compatible with the Ready-To-Run Separation Unit. The unit was designed to operate with premade TBE/ethidium bromide agarose cassettes and without additional liquid running uffer. The first gel, attempted on Saturday, reported "E1": overcurrent. Draining the TAE buffer caused an "open circuit" error. The only arrangement with which the machine initially appeared electrically content was the gel contacting only narrow segments of the bare electrodes at a 30 degree angle away from normal, a setup entirely incompatible with practical use.

The next idea was to cut the approximately 2.5"x4" gel into a 2.5"x2.5" square that would minimize electrode contact while keeping the charge gradient in the correct direction relative to the wells. This worked for a short while, but without TAE to maintain the gel, within a few minutes, the gel had shrunk enough to lose contact with the negative electrode. Even covering the gel surface with TAE again caused the "E1": over-current error.

A second 1.5% agarose/TAE gel was cast in the unit, after sealing some nooks (the areas beyond the electrodes, for instance) with electrical tape. Andrew Segina suggested a more conservative 30 mL from the 50 mL used in the first gel, but this volume was insufficient to entirely fill the curved surface of the plate. An additional 30 mL was prepared and overlaid on the first without issue. However, this gel suffered effectively the same issues as the first: without TAE, an undercurrent, with TAE, an overcurrent. Periodically filling the widening gaps between the electrodes and the gaps with additional strips of agarose gel helped prolong operation.

For testing, a sample of 2-log DNA ladder, a a sample of gel loading dye, and an opportune sample of 1% methylene blue (it was mentioned that it would stain DNA) were inserted into subsequent wells. The DNA ladder diffused quickly, the gel loading dye diffused more slowly and migrated in the correct direction, and the methylene blue migrated backwards to the negative electrode and began spilling into the unit's plate. Further research indicated that methylene blue held a positive charge and so migrated backwards. Andrew Buss discovered a protocol which revealed that methylene blue should be added after the gel run concludes.

A third gel was cast in the unit from 50 mL of mix. The intention was to allow the gel to entirely fill the plate, including behind the electrodes. When the gel had cooled, these areas were cut out with a razor and disposed of. The gel was lightly doused in TAE, as were the spaces between the gel and the electrodes. In this arrangement, the unit continued for nearly 5 minutes before it reported an undercurrent condition again.

Andrew Buss moved the gel to a mini gel box. The only available power supply had a range of 0-20V, so some previously inserted loading dye migrated very slowly. Tomorrow, it is hoped that we can borrow a benchtop power supply with a more reasonable output voltage range.

Vansh Singh and Evan Santos searched for and found the promoters, inducers, and reporters for the project.

Grant Hassinger started miniprep on his inoculated E. coli with parts A & B and attempted to pause the process through freezing at -20C

April 30

Andrew Buss obtained a 100/50 volt power supply designed specifically for electrophoresis. A gel loading dye was run alongside a DNA ladder for 50 minutes at 100 volts. The gel was then removed from the tray and soaked in 0.002% methylene blue at room temperature for 3 hours. Before the lab closed, the gel was left to wash in 1X TAE.

After Vansh Singh completed plasmid design, Andrew Buss used the Parts Registry to identify existing parts that would eliminate later time-consuming steps for the "rock" plasmid. First, an intermediate part by Randy Rettberg eliminates the assembly of an RBS and a cyan fluorescent protein. Another intermediate part eliminates the assembly of an RBS and a LuxR coding region. These reduce the number of required assemblies to three, which cannot be parallelized because of the requirement below that we produce the independent AraC sensor.

Andrew Buss documented the final and intermediate steps of assembly of the "rock" plasmid. First, we will construct a simple AraC sensor. Then we will assemble this sensor with a LuxR coding region to form our final plasmid. The intermediate AraC sensor will serve as a useful diagnostic tool, and will be submitted to the Registry as well.

Andrew Buss began to write a high-level protocol for assembly, detailing the restriction and ligation steps required to complete the plasmid.

Grant Hassinger resumed his miniprep but as the next step did not result in the desired effect, he concluded the cells should not be frozen

May 1

Andrew Buss's agarose gel was a uniform blue color, even after rinsing with TAE overnight. No bands were visible.

Andrew Buss transformed competent cells with the ligation from 4/29.

Andrew Buss resuspended DNA samples of BBa_R0080, BBa_B0010, BBa_J373033, and pSB1C3 from the Spring 2012 DNA distribution in 50 uL of distilled water each. Transformation is postponed until ampicillin media becomes available (ampicillin and other supplies are on order).