Difference between revisions of "Team:UCSC/Demonstrate"
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Revision as of 09:02, 1 November 2017
RESULTS
Our project focuses on biosynthetically producing the molecules vitamin B12 and acetaminophen in Synechococcus elongatus PCC 7942. The secondary focuses of our project was geared towards analyzing the growth of S. elongatus PCC 7942 in seawater, creating a detection method for vitamin B12 via a riboswitch/reporter system, and a proof of concept for the production of acetaminophen in E. coli.
I. Biosynthesis
1a. Plasmid construction of DWB1, DWB2, DWB3, and DWB4
Plasmids DWB1, DWB2, DWB3 and DWB4 were designed for gene integration into the chromosomal genome of S. elongatus PCC 7942. The plasmid AM2991 was constructed for homologous recombination into defined Neutral Integration Site 1 (NSI) in S. elongatus PCC 7492. The genes ssuE and 4ABH were assembled into a AM2991 plasmid backbone constructing DWB1 and DWB2, respectively. The plasmid AM1573 was used for recombination into Neutral Integration Site 2 (NSII). The genes bluB and nhoA were assembled into pAM1573 forming DWB3 and DWB4, respectively. All plasmids were successfully constructed and confirmed through sanger sequencing.
1b. Successful integration of ssuE into S. elongatus PCC 7942
Homologous recombination of DWB1 plasmid into the chromosomal genome of S. elongatus PCC 7942.
Colony PCR of a single cell line transformed with DWB1. Amplification of the first neutral integration site in S. elongatus PCC 7942 shows two bands: the original unmodified genome at ~1690bp and ssuE integrated cassette into the chromosomal genome at ~6500bp. An isolated cell culture with successful integration into most of the genome is seen on Patch #4.
Integration of the ssuE gene into S. elongatus PCC 7942 genome was successful. Cells were inoculated with DWB1 plasmid and transformed in the dark, overnight. Transformed cells were selected for growth on BG-11 and streptomycin plates. Colonies were grown for 7-10 days and homologous recombination of the entire plasmid cassette into the genome was assayed through PCR of neutral site one.
Due to multiple copies of the chromosomal genome in the organism, S. elongatus PCC 7942 was patched onto antibiotic plates after colony picks, with the purpose of isolating cells that contain solely integrated gene constructs. After four rounds of patching a single cell line, specific amplification of a strand larger than that of the original genome indicates successful isolation of ssuE integrated cells.
III. Growth
Growth curve of S.elongatus PCC 7942 cultures grown in SCCB-1 and BG-11. Absorbance with an OD730 was taken daily.
2a. S. elongatus grown in local sea water, SCCB-1
S. elongatus PCC 7942 cultures are commercially grown in BG-11 media. However, seawater from a local beach was obtained, SCCB-1, to deduce whether locally sourced bodies of water were sufficient for the growth of S. elongatus. SCCB-1 was filtered, autoclaved and supplemented with a source of Nitrogen in the form of NaNO2.
To show the difference in growth with each media, SCCB-1 and BG-11(N+) were inoculated with S. elongatus PCC 7942 and cell density was measured daily for 10 circadian days. Cultures grown in SCCB-1 exhibit similar growth patterns to that of BG-11(N+) confirming the potential of locally sourcing a media for cyanobacterial growth.
2b. Growth Rates and Selection
Growth Curves for Successive Inoculations of S. elongatus PCC 7942 at Various Wavelengths
Click here to view our growth data!
2c. Toxicity Tests
In order to judge whether our project would possibly be safe for consumption, we performed toxicity tests for the acetaminophen pathway in PCC 7942. Varying amounts of acetaminophen were added to liquid culture and the OD of each sample was observed. Liquid culture samples containing 1.32mM, 6.62mM, and 13.23mM of acetaminophen were left to oxidize overnight and the color change was compared with liquid cultures samples containing similar concentrations of 4-aminophenol.
Acetaminophen toxicity tests
4-aminophenol toxicity tests
Cell culture toxicity tests were performed with 4-aminophenol. Cell cultures were observed after the addition of 1.8µM, 9.1µM, and 18.3µM respectively. All samples with 4-aminophenol added turned from dark green to dark brown overnight.
A cell culture toxicity test suggested 13.32mM (10mg) of acetaminophen to be detrimental to cell growth. While the amount of time at which the cell culture showed discoloration from green to nearly clear to brown varied in duplicate samples, both concluded with cell death and discoloration of cell sample. Cell culture containing 6.6mM (5mg) of acetaminophen showed inconsistent discoloration and cultures containing 1.8mM (1mg) of acetaminophen showed no significant color change. Cultures were also tested with 1.8µM and 2.9µM of acetaminophen (to align with the expected amount based on a patent for the biosynthesis of acetaminophen in E. coli [ patent ] ). No significant discoloration was observed. The discoloration and subsequent color change of the culture is suspected to be due to the degradation of acetaminophen into its precursor 4-aminophenol. Further tests to determine the contents of the sample following the observed color change is recommended.
VI. Riboswitch
A detection mechanism for vitamin B12, specifically the molecule cobalamin, would allow for an instantaneous assay of its presence. The biobrick part, BBa_K1913011, from the Wageningen 2016 iGEM team was modified and tested for its affinity to 5,6-DMB-cobalamin (DMB B12) or adenine-cobalamin (B12 analogs). The part consist of a riboswitch, btub, that binds specifically to cobalamin, sequestering the ribosomal binding site (RBS) upstream of the TetR gene, thereby hindering the expression of the repressor. In the absence of cobalamin, the TetR gene is constitutively expressed producing the Tet repressor which trans-regulates the Tet operator, TetO. The operator was designed upstream of the reporter gene, mRFP. Therefore, detection of the reporter RFP implies the presence of vitamin B12.
Plasmid EN15 was constructed through Gibson Assembly. Gene block fragments consisting of the riboswitch/reporter parts, NSII homologous sequences, and an ampicillin resistance were ligated onto an E. coli specific sequence, pBR322. The novel plasmid was designed for recombination into Neutral Integration Site 2 (NSII) within the chromosomal genome of S. elongatus PCC 7942. The riboswitch system was modified for expression within S. elongatus PCC 7942. A light induced psbAI promoter, documented to be constitutively expressed in cyanobacteria such as S. elongatus PCC 7942, was placed upstream of the biobrick parts, producing two separate mRNA strands.
Expression of the plasmid EN15 produces two strands of mRNA. In the absence of cobalamin, the Tet repressor is expressed hindering the expression of the reporter gene. In the presence of cobalamin, mRFP thus becomes expressed.
The riboswitch system was modified to be expressed in E. coli as a proof that all parts work as expected. E. coli produces vitamin B12 analogs which bind onto the riboswitch and express the mRFP reporter gene. Once the assembly of the plasmid EN15 was confirmed, it was then transformed into E. coli which further validated that all parts worked accordingly. Plasmid EN15 was confirmed via sanger sequencing.
