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Revision as of 07:16, 3 October 2012

Penn State iGEM Notebook

Notebook

These are our day-to-day lab activities. Here you can see our successes, failures, and alterations in our plans as we continue to gain hands-on experience in the cutting-edge field of synthetic biology. The journey was long an arduous, but there is a light at the end of the tunnel, and that feeling when months of work pay off is worth every minute of disappointment.

Notebook

Week 1

Monday, May 22, 2012
We prepared ampicillin and streptomycin plates in preparation for production of plasmids and competent cells.

Tuesday, May 23, 2012
Inoculation of LB broth with DH10B E. coli strain to prepare master mix/culture of cells in preparation for making chemically competent cells. These cells were incubated throughout the day and prepared for competency by the end of the day. Promotes were also planned out for the Multiple Promoters Project and ordered. These promoters were designed to combine some of the components of the plasmid.

Wednesday, May 24, 2012
Biobricks were hydrated and transformed into Chemically Competent cells through heat shock ( BBa_R0063, BBa_R1062, BBa_R0078, BBa_I1051, BBa_R0079, BBa_R0071 ). LOB broth was also prepared for the recovery from the transformation. These cells were then plated to produce isolated colonies.

Thursday, May 25, 2012
Additional Biobricks were hydrated and transformed into Chemically Competent cells through heat shock ( BBa_I712074, BBa_R0062, BBa_I746104, BBa_I719005, BBa_J23119, BBa_J23100, BBa_J23101, BBa_J23102, BBa_J23103, BBa_J23104, BBa_J23105, BBa_J23106, BBa_J23107, BBa_J23110 ). LOB broth was used to help the cells recover from their transformation. More agar plates were poured. The Transformed cells form yesterday yielded isolated colonies. A single colony from each plate was used to create a broth in preparation for mini-prep of the plasmids.

Friday, May 25, 2012
Miniprep was done to extract the first six plasmids we transformed

Colonies that grew from the transformation of our last 14 plasmids were cultured for later plasmid extraction:

Saturday, May 26, 2012
Hannah and Kevin extracted the plasmids from the 14 colonies that were cultured yesterday. Two of the BioBricks that needed to be retransformed ( BBa_J23102, BBa_J23105) had isolated colonies on their plates and were cultured by Kevin for plasmid extraction the next day.

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Week 2

Sunday, May 27, 2012
Hannah and Kevin extracted the two cultures that Kevin prepared yesterday. These plasmids were put on ice for concentration analysis later with the other extracted plasmids.

Monday, May 28, 2012
Memorial Day

Tuesday, May 29, 2012
The 20 plasmids that have been grow during the previous week and weekend were digested by Hannah, Victoria, and Kevin. The plasmids were run in a gel, however errors in the gel's preparation resulted in a poor run. Always make sure your gasket is correctly seated before you pour the agarose!

Wednesday, May 30, 2012
Plasmids for the Multidirectional Promoters Project were transformed and cultured by Hannah and Kevin. Chris, Kait, and Victoria re-transformed 10 plasmids and cultured those. A gel was run to confirm our plasmids and fragments but was inconclusive due to low concentration.

Thursday, May 31, 2012
Fragments for the Multidirectional Promoter Project were digested and run through PCR by Hannah and Kevin. Kait and Victoria re-transformed the remaining 10 plasmids and 1 from yesterday that did not yield colonies. Kait and Victoria ran a gel to confirm a plasmid they could use in the Codon Optimization Project.
The originally transformed promoters were disposed of. Some of the cultures became a purple color. After centrifuging it was shown that the bacteria themselves were purple. This may be from contamination, so to be on the safe side, these plasmids were re-transformed. This may be an artifact from the plasmids themselves, but there is no consistent pattern. Very strange indeed! Always remember to use good sterile techniques when working with media and cultures!

Friday, June 1, 2012
Today Hannah digested components for the Multiple Start Codon Project. Later she ran a gel and extracted the components for later use. Kait and Victoria prepared a plasmid for the Codon Optimization Project. Kevin and Chris began work on a plasmid backbone and fluorescent component of the Multidirectional Promoters Project.
The team also received a new gel illuminator to see their bands without the use of UV light. They were very excited.

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Week 3

Monday, June 4, 2012
Hannah hydrated BBa_I13521 and used it to gauge the competency of the chemically competent cells we prepared earlier. Victoria and Kait ran a gel to confirm some of the components they need for the Codon Optimization Project. Kevin and Chris worked on some cleaning and housekeeping.

Tuesday, June 5, 2012
Victoria and Kait completed a transformation for the Codon Optimization Project. Kevin mini-prepped two plasmids for the Multidirectional Promoters Project. Chris and Kevin then froze some cultures containing these plasmids for later use. Hannah completed a CBAR and transformation for the Multiple Start Codon Project. She also tested the competency rate of the chemically competent cells the team prepared earlier.

Wednesday, June 6, 2012
Kevin and Chris ran a gel to separate components for the Multidirectional Promoters Project's final construct. Victoria and Kait inoculated cultures with isolated colonies. This culture has a plasmid that they will use for their final construct. Hannah is in the process of building a higher efficiency Chemically Competent DH10B cell line. The higher transformation rate will help not only the team but is critical for her CBAR assembly of her construct for the Multiple Start Codon Project. Hannah also started planning and development of a secondary construct for the Multidirectional Promoters Project to act as a control for the experiments.

Thursday, June 7, 2012
Kevin and Chris ran a gel to separate another component for the Multidirectional Promoters Project. One of the component did not run well on the gel. This component was amplified again with PCR. Kait and Victoria continued work on the Codon Optimization Project with the digestion of their plasmid, and subsequent gel extraction of the specific components. Hannah continued work on designing the primers for the CBAR reaction to create the control construct for the Multidirectional Promoters Project. Hannah also prepped a culture in an attempt to get a stock culture of DH10B growing. Her goal is to create a higher efficiency Chemically Competent (CC) DH10B cell line to help her complete her CBAR for her Multiple Start Codon Project.

Friday, June 8, 2012
Kevin extracted components for the Multidirectional Promoters Project. from the gel that was run yesterday. Hannah worked more on the design of the primers for one of the constructs for the Multidirectional Promoters Project. Kevin completed a streak plate to create isolated colonies in preparation for the production of more Chemically Competent cells. Kait and Victoria continued their work on their construct for the Codon Optimization Project. Victoria designed primers for their construct.

Saturday, June 9, 2012
Kevin and Chris produced a new set of Chemically Competent cells. Their goal is to create a cell line with a higher transformation efficiency.

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Week 4

Monday, June 11, 2012
Victoria and Kait mini-prepped a plasmid for their final construct. Hannah and Victoria poured new chloramphenicol plates. Chris ran a digest on six plasmids for the Multidirectional Promoters Project. Kevin and Hannah transformed six plasmids for this project as well. Kevin ran a transformation to test the competency of the cells that he and Chris prepared over the weekend.

Tuesday, June 12, 2012
Hannah completed her CBAR transformation today. Kait, Victoria, and Alexander completed a gel, gel extraction, and transformation for constructs for the Codon Optimization Project. Kevin and Chris inoculated six cultures with transformed colonies that grew overnight. Chris also did a comparison transformation between the CC DH10B cell line that he and Kevin made over the weekend, and the other CC cells that the team has been using.

Wednesday, June 13, 2012
Victoria and Kait made more plasmid stock for the Codon Optimization Project. Hannah made cultures from the colonies that grew from her CBAR reaction. Kevin and Chris extracted plasmids for the Multidirectional Promoters Project. They also began making more plasmid vector in preparation for their CBAR reaction.

Thursday, June 14, 2012
Plasmids from Hannah's CBAR reaction were extracted and back-up cultures were inoculated. Victoria, Kait, and Alexander ran a gel to confirm the components of plasmids to be used in the final Codon Optimization Construct. Kevin and Chris inoculated a culture with a colony containing the vector plasmid for the Multidirectional Promoters Project. They came in later that night to extract the plasmid.

Friday, June 15, 2012
Kevin ran a PCR on the vector component that he and Chris extracted the previous night. This was run through gel electrophoresis to separate the desired component from other side-products, and to ensure the fragment was the correct size. This was finally cleaned and concentrated for a CBAR reaction on Monday. Hannah grew her cultures from her CBAR reaction to get a higher plasmid yield. She also prepared new chemically competent DH10B cells since the stock was running low. Victoria, Kait, and Alexander checked a plasmid's components for the Codon Optimization Project.

Saturday, June 16, 2012
Hannah mini-prepped cultures for the Codon Optimization Project, and made TSS and Chemically Competent cells.

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Week 5

Sunday, June 17, 2012
Hannah came in and transformed the new Chemically Competent cells to test their competency. She also swapped the broth in her CBAR cultures to facilitate better growth.

Monday, June 18, 2012
Victoria prepared and stored cultures containing plasmids for the Codon Optimization Project. Hannah and Kevin extracted plasmids from colonies from Hannah's CBAR reaction. These were then digested and run on a gel. Kevin and Chris ran a PCR to produce a component for the final construct for the Multidirectional Promoters Project. The first reaction did not yield the expected results. Two more were prepared for comparison.

Tuesday, June 19, 2012
Hannah and Kevin extracted plasmids from the CBAR cultures again to get a higher yield for sequencing. Victoria and Alexander worked on the Codon Optimization construct by culturing insert components. They also extracted plasmids with the desired components for the final construct. These components were digested, run in a gel, extracted, and Cleaned and Concentrated. Kevin and Chris ran a gel to compare the PCR runs set up yesterday. This showed that the PCR runs had not yielded the desired components. Another PCR run was prepared and run a lower temperature for better annealing. Hannah ran a gel of her CBAR plasmid digests to see if they contained the correct parts. Unfortunately both gels showed that the plasmids were self ligations of a single component of the construct.

Wednesday, June 20, 2012
Victoria developed a new method for the assembly of the Codon Optimization Project. Hannah designed primers for the main construct for the Multiple Start Codons project. Kevin and Chris ran gels and extracted components for the final construct for the Multidirectional Promoters Project. They also ran another set of PCR to make more of the three components for the CBAR assembly they will need to do. Kait and Alexander extracted plasmids containing components for the Codon Optimization Construct.

Thursday, June 21, 2012
Kait and Victoria sent a construct for sequencing. Hannah compared the effects of a more concentrated LB broth on the growth of DH10B. Kevin completed a storyboard for an animation describing one of the projects. He also isolated components from the PCR runs set up yesterday through the use of gel electrophoresis.

Friday, June 22, 2012
Victoria, Kait, and Alexander annealed components for inserts for their project. Hannah also finished preparations for a new line of TSS CC DH10B.

Saturday, June 23, 2012
Kevin came in and ran a CBAR reaction on the components for the Multidirectional Promoters Project.

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Week 6

Monday, June 25, 2012
Kevin transformed the CBAR product into DH10B and plated it. Hannah worked on developing a document that condenses all of the protocols used in the lab. Victoria worked to develop new primers for building the main construct for the Codon Optimization Project. Chris and Hannah began work on new Chemically Competent DH10B cells. Hannah completed PCR runs for her new primers that arrived today.

Tuesday, June 26, 2012
The transformation from the CBAR reaction Kevin did yesterday proved to be successful, and produced many colonies. Six of these were chose and cultured. They will be tested to see if they contain the complete construct for the Multidirectional Promoters Project. Victoria, Kait, and Alexander worked on ligating components for the Codon Optimization Project. They also grew cultures containing components they would need later on in the assembly. Hannah ran PCR on some new segments of her final construct that just came in. These were later run in a CBAR. This new set of components would be better and produce the desired construct.

Wednesday, June 27, 2012
The cultures from the CBAR for the Multidirectional Promoters Project were mini-prepped and tested to see if they contained the correct components for the final construct. The gel to check this unfortunately did not set correctly and the cultures needed to be redone. Fortunately, Kevin had made a backup. Some additional colonies were also cultured to expand the search. Hannah worked on the sequencing primers for the Multidirectional Promoter construct. She also continued work on a document combining all of the protocols we use in the lab. Kait and Victoria completed digestions and ligations for the construction of their construct for the Codon Optimization Project.

Thursday, June 28, 2012
Cultures from the Multidirectional Promoters Project CBAR reaction were mini-prepped, again. 4 additional colonies were prepped to expand the search for the correct construct. These were then digested in preparation for use in a gel to confirm length. Hannah ran PCR on components for the Multiple Start Codon construct. She also inoculated cultures with colonies from one of her new CBAR reactions. Victoria, Kait, and Alexander worked on their own PCR runs to make the new Codon Optimization construct.

Friday, June 29, 2012
Hannah ran another PCR for one of her new G-blocks. She then mini-prepped cultures from her CBAR reaction. Some of the colonies seemed to be red, indicating they had taken up a plasmid with at least some of the desired components. Kait and Alexander ran gels to digest components and PCR products in preparation for an assembly of the construct for the Codon Optimization Project.

Saturday, June 30, 2012
Hannah completed a digestion on her CBAR plasmids and ran a gel to confirm their length. Chris ran gels to determine which colonies to have sequenced from the CBAR reaction for the Multidirectional Promoters Project. Kevin completed the beginning of an animation for the team.

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Week 7

Monday, July 2, 2012
Kevin completed another component for an animation. Hannah ran a gel to assess the length of the plasmids in the colonies she cultured from her CBAR reaction for the Multiple Start Codon Project. To test the stability of some of her plasmids, she transformed them back into DH10B cells. Chris grew up cultures with the desired colonies for sequencing to ensure there was enough sample.

Tuesday, July 3, 2012
Chris mini-prepped cultures to get plasmids for sequencing. Hannah inspected her cultures for their RFP expression, which was present. She used these plates to culture broth in preparation for sequencing. Victoria and Kait looked through some of the teams stocks for primers that could be used for sequencing the construct for the Codon Optimization Project. Sequencing primers were acquired for the Multidirectional Promoters Project.

Wednesday, July 4, 2012
Hannah inoculated cultures for the Multiple Start Codon Project. Victoria checked her plates for signs of expression of mCherry. The broth cultures for mini-prepping and sequencing still required another day of growth. Kevin made some progress with an animation for the team.

Thursday, July 5, 2012
Chris mini-prepped cultures for sequencing, but the yields were still low. Victoria, Kait, and Alexander mini-prepped their cultures in preparation for sequencing. Hannah also ran some mini-preps to prepare samples for sequencing. Kevin worked more on the animations and was shown a better way for making Chemically Competent cells.

Friday, July 6, 2012
Samples were sent off for sequencing for the Codon Optimization Project and Multidirectional Promoters Project. Updates were also made to the website concerning the Navigation Bar.

Saturday, July 7, 2012
Kevin came into the lab to escape the heat and spent some time working on an animation for the team.

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Week 8

Monday, July 9, 2012
Victoria, Kait, and Alexander ran digestions to check the length of some of their fragments. Chris check his cultures for growth, but they were not ready for mini-prepping yet. Kevin completed the first 93 seconds of an animation for the team.

Tuesday, July 10, 2012< br/> Chris received his sequencing data. It looked promising, however parts of it seemed inconclusive. He cultured this strain in preparation for testing it by inserting a promoter. Victoria ran a PCR reaction for some of the components for the Codon Optimization Project. Victoria and Alexander worked to make master cultures of components for the Codon Optimization Project.

Wednesday, July 11, 2012
Victoria, Kait, and Alexander mini-prepped cultures in preparation for sequencing. Chris transformed a plasmid, BBa_J23102, to use to test a construct that looks positive for having all the parts from the CBAR. Victoria also ran a digestion on the plasmids she prepared earlier to see if they possessed the correct restriction sites. The plasmids that looked promising were sent to sequencing. Kevin made more progress with the animation. He also worked with Nikki to help plan out the flow of the website.

Thursday, July 12, 2012
Chris checked his results from sequencing. With help from Hannah, he was able to conclude that there was a good chance that the plasmid was the desired construct. Hannah also checked her results from sequencing plasmids from her colonies from her second CBAR reaction. Her results showed to be negative, in response she ran another CBAR. Chris mini-prepped the J23102 that he grew up yesterday in preparation of inserting it into his suspected construct. Victoria ran a PCR for a fragment of the Codon Optimization Project, and cleaned the product of a previous PCR. Kevin made progress and created all new home and team pages. He also managed to get the navigation bar working, albeit in a crude format.

Friday, July 13, 2012
Chris checked the concentration of some of his mini-preps in preparation for sequencing and ligations. Victoria transformed some plasmids to be able to check them later for the desired parts. Victoria worked with Jyotsna to find some of the needed parts for the Codon Optimization Project. Kevin updated some of the webpages and poured some plates.

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Week 9

Monday, July 16, 2012
Chris ran some mini-preps for the Multidirectional Promoters Project. Victoria revved the results of her sequencing with Kait. Kevin worked with Hannah and taught her some HTML.

Tuesday, July 17, 2012
Hannah came in early to mini-prep cultures from her most recent CBAR for the Multiple Start Codon Project. These were subsequently digested to check for proper length. The result of this gel raised some questions. The colonies that she had grown up were all expression RFP. There were some colonies that were expressing GFP, but these were not cultured. Analysis of the gel and expression of RFP indicates that those plasmids did not contain all of the correct components. The GFP colonies probably would contain the entire construct. These were cultured to test this hypothesis. Victoria worked with Jyotsna to design primers to make some of the necessary parts for the Codon Optimization Project construct. Alexander is also beginning presentations for describing all of the projects. Kevin made some headway with the animation, which is till humming along. He also prepared materials for making new Chemically Competent cells.

Wednesday, July 18, 2012
Kevin updated some of the website and made some new chemically competent cells. Hannah and Kevin then transformed a number of fluorescent protein plasmids into these new cells. Hannah prepped her GFP presenting colonies from her CBAR. These were then digested to check them for length. The correct plasmids were prepped to be sent to sequencing. Victoria planned and designed primers for the Codon Optimization Project.

Thursday, July 19, 2012
Victoria ran a PCR of one of her components for the Codon Optimization Project. Hannah sent her plasmids to sequencing. Some of the plasmids that she transformed yesterday with Kevin were successful and presenting the correct fluorescent protein. Chris ran a gel on the construct for the Multidirectional Promoter Project. Kait and Victoria transformed some plasmids in preparation for another PCR to make a fragment for the Codon Optimization Project.

Friday, July 20, 2012
Victoria and Alexander completed a ligation to hopefully give them their desired construct. Kevin created a new run of chemically competent cells for the lab. Hannah ran a PCR of one of the components of her construct. This was to test to see if the deletion in the construct was caused by the PCR or if the problem resided somewhere else. Victoria also completed a digestion of a plasmid to check its length.

Saturday, July 21, 2012
Hannah transformed and plated her partial construct to have it as a back-up to work from to insert the deleted piece.

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Week 10

Sunday, July 22, 2012
Hannah came in to make cultures from isolated colonies from the plates she made yesterday.

Monday, July 23, 2012
Chris completed mini-preps and digestions on components to be ligated together to test the suspected construct for the Multidirectional Promoter Project. Kevin worked more on the animation and got to another milestone.

Tuesday, July 24, 2012
Hannah ran a PCR on the vector component for the Multiple Start Codon Project. She proceeded to complete another CBAR to try and get the missing segment into the construct. Kait and Victoria inserted a fragment into their vector backbone. This will hopefully yield the construct for the Codon Optimization Project. Kevin ran a gel extraction and cleanup on a gel run by Chris.

Wednesday, July 25, 2012
Chris completed some annealings for inserts for his construct for the Bidirectional Promoter Project (formerly known as the Multidirectional Promoter Project). Kait and Victoria ran some miniprepps and plated some cultures to create stocks of their construct components for the Codon Optimization Project. Hannah learned more about working with webpages. Kevin made some more progress with the animation.

Thursday, July 26, 2012
Chris completed some transformation into new CC and old CC. This will not only show if his construct is correct, but also determine how good the new CC are. Hannah ran some transformations to see if she got the completed construct for the Multiple Start Codon Project. Victoria tracked down the required materials for running flow cytometry on samples when the Codon Optimization Construct is completed. Kevin made more headway with the animation.

Friday, July 27, 2012
Kait completed 12 mini-preps of two components needed for one of the assembly methods for the Codon Optimization Project's construct. She also made some stock cultures for the freezer for long-term storage. She also picked some colonies and cultured them. Kevin continued work on the animation.

Saturday, July 28, 2012
Kevin came in to check on some transformations Chris had plated. Unfortunately, all of the plates showed no growth.

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Week 11

Sunday, July 29, 2012
Kait came in and plated some cultures for the Codon Optimization Project. These cultures will hopefully yield the desired functioning construct with the new promoter.

Monday, July 30, 2012
Kait completed a ligation and transformation for the Codon Optimization Project. Chris completed a number of mini-preps for the Bidirectional Promoter Project. Kevin made more progress with the animation.

Tuesday, July 31, 2012
Kait completed mini-preps for the Codon Optimization Project to create more stocks for later. Chris completed another transformation of his ligated component.

Wednesday, August 1, 2012
Kait completed a ligation and transformation using one of the other methods for synthesizing the desired construct for the Codon Optimization Project. Kevin completed some preparations for sequencing some colonies from a CBAR for the Multiple Start Codon Project. He also continued work on the animation.

Thursday, August 2, 2012
Kait and Chris both reran ligations and transformations after the identification of an error in the procedure. Kevin continued work on the animation and prepared more media for the team.

Friday, August 3, 2012
Kait completed a mini-prep for the Codon Optimization Project. Chris completed a six hour ligation. Kevin worked one the animation and prepped samples for sequencing for the Multiple Start Codon Project. He also ran tests to check the efficiency of the competent cells he made earlier.

Saturday, August 4, 2012
Another milestone was reached in the animation. It is almost done!

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Week 12

Sunday, August 5, 2012
Chris checked on his digestion from Friday, and completed a ligation.