Team:UIUC Illinois/Project
Background
Gibson Assembly is a revolutionary method for assembling multiple linear DNA fragments (original paper link here) by Dr. Daniel Gibson at the J. Craig Venter Institute in 2009. The multiple overlapping DNA fragments can be joined by a single reaction regardless of the fragment length, which adds to the versatility of the method. By adding the three different enzymes (5’ exonuclease, DNA polymerase, and DNA ligase), a fully ligated double-stranded DNA molecule is acquired. This method is proven to be efficient due to the ease of the reaction – needing only one tube of reaction – and the effectiveness of the reaction: no scars on the ligated DNA, non-selective compatibility of DNA fragments, and no specific restriction sites are needed.
- Primer Design In designing the primer, adjacent segments in the plasmid should have identical sequences on the ends; the insert sequence and the vector sequence should be compatible with each other. These identical sequences can be created via PCR with primers containing a 5’ end identical to the adjacent segment and a 3’ that would anneal to the target sequence. Also, an effective amount of 60 bp primers might be more effective due to the more targeted approach by the enzymes. Once a preferred primer design has been achieved, amplify the amount of primer DNA by PCR.
- Check the purity and concentration of the PCR amplification and/or restriction digest. If the product is found to be impure, perform a gel purification to remove impurities.
For our method, we used NEB’s Gibson Assembly Master Mix. The Master Mix consists of three enzymes in a single buffer:
- 1. T5 exonuclease: chews back the 5’ end of the DNA to create a 3’ overhang, so the complementary strand would anneal to each other.
- 2. Phusion DNA Polymerase: incorporates nucleotides to fill in the gaps in the annealed DNA fragment
- 3. Taq DNA ligase: joining the annealed DNA fragments and removing the ‘nicks’ and ‘scars’.
The method can simultaneously combine up to 15 DNA fragments based on sequence identity. It requires that the DNA fragments contain ~20-40 base pair overlap with adjacent DNA fragments. The appropriate amount of DNA, when combining 2-3 fragments in a Gibson Assembly reaction, is 0.02 – 0.5 pmol of total DNA. Cloning efficiency is best when 50 -100 ng of vector is used, with 2-3 equivalents of insert in a 20 µl reaction. Sample should then be incubated in a thermocycler at 50ºC for 15 minutes, then sample should be saved in –20ºC until further examination is made.
Experiment Description
Once of the biggest obstacles to scientific advancement and discovery is the exorbitant monetary price for conducting small experiments. To put this in perspective, the purchase of one Gibson Assembly Kit for ten reactions is currently $185.00. For research that heavily depends on assembling different DNA fragments, the price can rise very quickly.
The aim of creating a homemade Gibson Assembly recipe was to drastically lower the prices of performing Gibson Assembly. The price could be lowered if the same outcome could be performed using unpurified enzymes. The unpurified enzymes would replace the purified enzymes commonly used in the Gibson Assembly master mix.
The inspiration for this project stems from Genentech’s 1978 success story when the first successful production of human insulin in the laboratory was announced[1].
Scientists at Genentech used recombinant DNA techniques to produce a hybrid DNA molecule that contained the gene to produce human insulin. This recombinant plasmid was inserted into E. Coli cells for mass production of the human insulin protein hormone. Thus, the underlying process of our experiment is the same . However, we used a common recombinant DNA technique to our advantage.
The idea of our project can be summarized in one sentence “using Gibson assembly to make enzymes for Gibson Assembly.”
To expand on this statement, the Gibson assembly cloning method was used to create constructs that contained the genes for DNA polymerase and DNA ligase. These constructs were created so that when they are transformed into DH5α cells, the local cell machinery would be used for high expression of the gene and produce an abundant number of DNA ligase and DNA polymerase enzymes.
Once colonies of the transformed cells grow, cell lysate will be made in buffer similar to the Gibson assembly buffer. Two tubes of cell lysate, one containing DNA ligase and the other containing DNA polymerase, will be added in ratios relative to each other to the reaction tubes. The ratio that yields results will be determined empirically. The background exonuclease naturally present in the lysate would account for the exonuclease needed for the reaction.
To verify that the isothermal reaction using unpurified enzyme worked, we transformed the allegedly assembled construct and then purify the DNA from the bacterial colonies. This purified DNA is sent for sequencing. If the returned matches the known sequence, we conclude that the isothermal reaction was successful.
[1] Press Releases. (1978, September 6). Retrieved October 29, 2017, from https://www.gene.com/media/press-releases/4160/1978-09-06/first-successful-laboratory-production-o
