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), TdT delta26-143 (BBa_K1746001), and TdT GIP 213-215 subAAA (BBa_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
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
June 30th
Tushar, Chris, Keith, Susung, John, Jean
Sent in pSB1C3 + TdT variant colonies to be sequenced
JULY 2015
July 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.
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.
Ben, Susung
Sourced parts
AUGUST 2015
August 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
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
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 2015
September 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
