iGEM 2015 — Team Cooper_Union

# Team:Cooper Union/Notebook

Cooper Union 2015 iGEM

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# Cooper Union iGEM 2015 Notebook

## JUNE 2015

#### June 3rd - 8th

Tushar, Chris, Keith, Susung, John, Jean

- We designed various truncated and modified TdT sequences using the SnapGene viewer. The sequences were derived from the results documented in a paper by Repasky et. all detailing the Mutational Analysis of Terminal Deoxynucleotidyltransferase-mediated N-Nucleotide Addition in V(D)J Recombination.
- The following sequences were designed: TdT delta1-27 [(BBa\_K1746000)](http://parts.igem.org/Part%3ABBa_K1746000), TdT delta26-143 [(BBa\_K1746001)](http://parts.igem.org/Part%3ABBa_K1746001), and TdT GIP 213-215 subAAA [(BBa\_K1746002)](http://parts.igem.org/Part%3ABBa_K1746002).

#### June 16th

Tushar, Chris, Keith, Susung, John, Jean

- We practiced making 1% agarose gels. We immobilized DNA to Dynabeads (following the Dynabead Prep Protocol) and then ran experiments with TdT using regular dATPs and Cleanamp dATPs (following the Dynabead Cleanamp Protocol). We ran a gel of the experiments but saw no results. It is proposed that DNA was lost when removing DNA from Dynabeads.

#### June 17th

Tushar, Chris, Keith, Susung, John, Jean

- Prepared more Dynabeads with immobalized DNA (following the Dynabead Prep Protocol)
- Prepared LB Broth
- Prepared more 1% agarose gels

#### June 18th

Tushar, Chris, Keith, Susung, John, Jean

- Ran gels of Cleanamp Dynabead Experiment, but saw no results.

#### June 22nd

Tushar, Chris, Keith, Susung, John, Jean

- To test our polyacrimide gels (they were kind of old) we ran another polyacrimide gel with regular DNA sequences
![](https://static.igem.org/mediawiki/2015/3/3b/CooperUnion_Notebook_6-22.jpeg)

#### June 25th

Tushar, Chris, Keith, Susung, John, Jean

- Received the TdT del1-27. del26-143, and GIP subAAA variants!
- Did a double digest of TdT variants and pSB1C3 ADD PROTOCOL
- PCR purified restriction enzyme digest products and then performed a ligation with T4 DNA Ligase ADD PROTOCOL

#### June 26th

Tushar, Chris, Keith, Susung, John, Jean

- Performed a Transformation of TdT variants cloned into pSB1C3 with NEB5alpha cells

#### June 29th

Tushar, Chris, Keith, Susung, John, Jean

- Made two 1% agarose gels
- Observed that colonies grew on our transformation plates
- Did a colony PCR of multiple colonies
- Ran PCR products on a gel
- Made backup colonies for cells that had vector + insert
![](https://static.igem.org/mediawiki/2015/7/73/CooperUnion_Notebook_6292.jpeg)
![](https://static.igem.org/mediawiki/2015/b/bf/CooperUnion_Notebook_6294.jpeg)
![](https://static.igem.org/mediawiki/2015/5/56/CooperUnion_Notebook_6296.jpeg)

#### June 30th

Tushar, Chris, Keith, Susung, John, Jean

- Sent in pSB1C3 + TdT variant colonies to be sequenced
![](https://static.igem.org/mediawiki/2015/8/85/CooperUnion_Notebook_630.jpeg)

## JULY 2015July 1st Tushar, Chris, Keith, Susung, John, Jean - Followed Double Digest Protocol for Pet28b+ with BamHI and XbaI; this includes the double digest of Pet28b+ and TdT del 1-27. del 26-143, and GIP sub AAA TdT mutants - Did PCR purification - Really low concentration and bad graph on nanodrop - Was recommended to use less EB buffer in the future>- Reran digestion of Pet28b+ - Did PCR purification with less EB buffer - Performed a ligation of Pet28b+ and TdT variants using T4 DNA Ligase July 2nd Tushar, Chris, Keith, Susung, John, Jean - Did a transformation of ligations of Pet28b and TdT variants July 6th Tushar, Chris, Keith, Susung, John, Jean - Performed inoculations of certain colonies July 7th Tushar, Chris, Keith, Susung, John, Jean - Mini prepped inoculations from previous night - Nanodrop of colonies indicated good results and sent the samples to be sequenced July 8th Tushar, Chris, Keith, Susung, John, Jean - Sequencing results indicated no priming for samples, and therefore ligation did not work or the DNA was lost in some process afterwards July 9th Tushar, Chris, Keith, Susung, John, Jean - Re-ran double digests of Pet28b+ and TdT variants - Performed a gel purification of the digests and set up an overnight ligation of TdT variants + Pet28b+ July 10th Tushar, Chris, Keith, Susung, John, Jean - Made fresh LB Agar + Kanamycin plates - Transformed ligations from July 9th onto fresh Kan-plates July 14th Tushar, Chris, Keith, Susung, John, Jean - Inoculated colonies of TdT variants + Pet28b+ Ben, Susung - Discussed possible designs for the hardware. Marked out design goals as well as limitations. July 15th Tushar, Chris, Keith, Susung, John, Jean - Did Colony PCR of TdT variants + Pet28b+ colonies - Ran a gel of colony PCR, but results came out negative Ben, Susung - First draft of fluidics system using Tygon-tubing and solenoid valves July 17th Tushar, Chris, Keith, Susung, John, Jean - Planned the immobilization of disulfide modified oligos to mercaptosilane treated glass slides - Designed a disulfide oligonucleotide for immobilization and ordered 3-mercaptopropyl trimethoxysilane Ben, Susung - Design the initial thermocycling system using a series of resistors and switches - Gut a broken PCR machine for thermoelectric coolers July 20th-August 17th Chris, Keith, Susung, John, Jean worked on STEM Outreach Tushar worked on glass slide immobalization - During this time period, we spent most of our time working on our [High School Summer STEM Outreach](https://2015.igem.org/Team%3ACooper_Union/Practices). - We also spent our time perfecting our technique for creating mercaptosilane treated glass slides, and immobilizing disulfide modified DNA to them. This process involved a collaboration with the [Genspace iGEM Team](https://2015.igem.org/Team%3ACooper_Union/Collaborations). Ben, Susung - Sourced parts AUGUST 2015August 17th Tushar, Chris, Keith, Susung, John, Jean - Set up another double digest of Pet28b+ and the TdT variants with XbaI and BamHI Ben, Susung, Karlin - Initial fluidic system ran into problems - Source parts for peristaltic pump as alternative fluid system - Thermocycling parts arrived and being constructed - Gained access to 3D printer, and began printing peltier case August 18th Tushar, Chris, Keith, Susung, John, Jean - Performed a PCR cleanup of the double digests the Pet28b+ and TdT variants - Set up an overnight ligation of Pet28b+ and TdT variants - Set up an RNA ligase reaction of PNK treated 43mer and untreated 52mer Ben, Susung, Karlin - 3D print complete. First prototype of thermal cycler completed August 19th Tushar, Chris, Keith, Susung, John, Jean - To verify if ligation worked, instead of transforming immediately, performed PCR of ligation product - Ran an agarose gel of the PCR products, results were slightly conclusive; performed transformations of ligations of TdT variants + Pet28b+ - Ran a polyacrimide gel of RNA ligation, and results were also inconclusive Ben, Tushar - Met with Dac Anh in discussing the potential of moving to microfluidics as an alternative - Discussed advantages, disadvtantages, and feasibility - Concluded that it would be better to work from macro-size and scale down Ben, Susung, Karlin - Peristaltic pump parts arrive - First pump constructed and tested August 20th Tushar, Chris, Keith, Susung, John, Jean - Performed colony PCR of 5 colonies from each plate (5 from TdT del1-27 + Pet28b+; 5 from TdT del26-143 + Pet28b+; 5 from TdT GIP subAAA + Pet28b+) - Results were positive! Indicated TdT del1-27 inserts in Pet28+ - Made backup colonies of samples that had TdT del1-27 inserts ![](https://static.igem.org/mediawiki/2015/2/22/CooperUnion_Notebook_820.png) ![](https://static.igem.org/mediawiki/2015/d/de/CooperUnion_Notebook_8201.png) Ben, Susung, Karlin - Concerns raised about thermal cycler - Design a better system and order parts - Begin machining necessary components August 24th Tushar, Chris, Keith, Susung, John, Jean - Performed more colony PCR of the same plates from August 20th, to verify if any other colonies had inserts - Ran gel of colony PCR and verified cells with inserts containing TdT del26-143 - Made backup colonies of samples that had TdT del26-143 inserts - Inoculated previous colonies containing TdT del1-27 inserts ![](https://static.igem.org/mediawiki/2015/1/1f/CooperUnion_Notebook_8241.png) Ben, Susung, Karlin - Fried a motorshield - More motors and shields are ordered August 25th Tushar, Chris, Keith, Susung, John, Jean - Mini-prepped inoculated TdT del1-27 colonies and sent plasmid to be sequenced - Inoculated previous colonies containing TdT del26-143 inserts August 26th Tushar, Chris, Keith, Susung, John, Jean - Sequencing results verified Pet28b+ plasmids containing the TdT del1-27 inserts with an accuracies of over 97%! - Prepared and sent previous TdT del26-143 samples for sequencing August 27th Tushar, Chris, Keith, Susung, John, Jean - Sequencing results verified Pet28b+ plasmids containing the TdT del26-143 inserts with an accuracies of over 98%! - Transformed all good TdT variants into Rosetta Protein Purification Cells Ben, Susung, Karlin - New parts arrive - New fluid system tested - New thermal cycler constructed SEPTEMBER 2015September 2nd Tushar, Chris, Keith, Susung, John, Jean - Inoculated Rosetta cells containing Pet28b+ with ligated TdT del1-27, and TdT del26-143 September 3rd Tushar, Chris, Keith, Susung, John, Jean - Used some inoculate to make backup colonies of Pet28b+ with ligated TdT del1-27, and TdT del26-143 - Mini-prepped Pet28b+ with ligated TdT del1-27, and TdT del26-143 - Designed and ordered a new reverse ligation oligo for RNA ligation reaction Ben, Susung, Karlin - Program Arduino to operate motors and thermal cycler September 8th Tushar, Chris, Keith, Susung, John, Jean - Set up RNA ligation with new reverse ligation oligo Ben, Susung, Karlin - Extra subsequent pieces are machined and printed - Subsystems are all functional September 18th Tushar, Chris, Keith, Susung, John, Jean - Ran a gel of RNA ligation from September 8th, results were promising. It seems as if there could be spill over from other gels - View our [results](https://2015.igem.org/Team%3ACooper_Union/Results) for an in-depth analysis of these RNA Ligase reaction results ![](https://static.igem.org/mediawiki/2015/0/02/CooperUnion_Notebook_9181.jpeg)