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

Andrew Segina	LB plate	Part A from kit	Ampicillin, kanamycin
Andrew Segina	LB plate	Part B from kit	Ampicillin, kanamycin
Andrew Buss	SOB plate	NEB 10 from kit
Andrew Segina	SOB plate	NEB 10 from kit
Andrew Buss	LB tube	Part A from kit		Incubating in dry bath
Andrew Buss	LB tube x2	Part A from kit		Incubating in dry bath
Andrew Buss	LB tube x2	Part B from kit		Incubating in dry bath
Vansh Singh	LB tube	Part A from 4/10 plate		Incubating in dry bath
Vansh Singh	LB tube	Part B from 4/10 plate		Incubating in dry bath
Andrew Buss	LB tube	NEB 10 from 4/10 SOB plate		Incubating in dry bath

April 13 available media

Andrew Segina	800 mL YPD	Lacks dextrose
Andrew Buss	4 YPD plates
Andrew Buss	2 LB plates
Andrew Segina	8 LB plates
Andrew Buss	~10mL LB tube	In dry bath for comparison
Andrew Buss	~40mL LB broth
Vansh Singh	~5mL LB broth	Ampicillin, kanamycin
Grant Hassinger	100mL LB broth

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

Andrew Segina	LB plate	Part A from kit	Ampicillin, kanamycin	In refrigerator
Andrew Segina	LB plate	Part B from kit	Ampicillin, kanamycin	In refrigerator
Andrew Buss	SOB plate	NEB 10 from kit		In refrigerator
Andrew Segina	SOB plate	NEB 10 from kit		In refrigerator
Andrew Buss	LB tube	Part A from kit		Incubating in dry bath
Andrew Buss	LB tube	Part B from kit		Incubating in dry bath
Andrew Buss	LB tube	Part A from 4/10 plate		Incubating in dry bath
Andrew Buss	LB tube	Part B from 4/10 plate		Incubating in dry bath
Andrew Buss	LB tube x3	NEB 10 from 4/10 SOB plate		Incubating in dry bath
Andrew Buss	LB plate	NEB 10 from 4/10 SOB plate		Incubating
Andrew Buss	LB plate	NEB 10 from 4/18 NEB 10 plate		Incubating

April 20 available media

Andrew Segina	800 mL YPD	Lacks dextrose
Andrew Segina	8 LB plates
Andrew Buss	~5mL LB broth
Vansh Singh	~5mL LB broth	Ampicillin, kanamycin
Grant Hassinger	100mL LB broth
Grant Hassinger	100mL LB broth

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.