Team:Nevada/Notebook/Weeks14

Calender Weeks 1-4
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Week 1 - June 1st-5th

E. Coli

Lab clean-up, media preparation. Registry distribution received from iGEM Headquarters. Prepared DNA from iGEM kit for future use (pSB1C3, pSB1A3, pSB1K3 and σ70 constitutive promoter).

Bay Laurel Thioesterase

MT:Codon optimized the Bay Laurel Thioesterase for E. coli. Ordered gene through gene script with RBS and terminator.

Cyano
Lab preparation. Equipment transport and removal.

Enzymology
Hexokinase coupled assay used to quantitate amounts of cyanobacteria glucose secretion.

Rxn:
• 1. glucose + ATP --Hx-> G-6-P + ADP
• 2. G-6-P + NAD+ --G-6-PDeH-> G-6-P + NADH

Reaction used Glucose assay kit (Genzyme) containing 2000U/L Hexokinase reagent and 4000U/L G-6-P DeH and measured glucose concentrations ranging from 0.05mM-0.25mM and absorbances were measured at 340.0 nm. Results: Final NADH concentrations were determined using the molar extinction coefficient and were proportionate to initial glucose concentrations. Discussion: Focus is now to measure not only glucose, but fructose concentrations as well as both with be secreted in the form of sucrose using a D-glucose/D-fructose assay (Megaenzyme).

Media
Formation of Media Group.

Week 2 - June 6th-12th

E. Coli

Transformed iGEM plasmids (pSB1C3, pSB1A3, pSB1K3) and σ70 constitutive promoter (J23101) into NEB10 β strain of E. coli (New England Biolabs). Inoculated single colonies in LB-Ampicillin to create liquid cultures. Performed minipreps and nanodrop analysis of cultures.

To confirm products, 0.5ug of DNA was digested with EcoRI. Digestions were run on a 1.2% gel, and bands obtained confirmed pSB1C3 (2070bp), pSB1A3 (2155bp), pSB1K3 (2204bp), and σ70 constitutive promoter (35bp).


Cyano

Procedure: Synthetic genes encoding for the GLF protein and invertase were ordered and transformed into DH5α cells. Transformants were selected for on ampicillin plates and cultured in LBamp broth. Plasmid DNA from the transformants was isolated using a QIAgen miniprep kit and submitted for sequencing.

Results/Discussion: Sequencing data indicated successful transformations of DH5α cells with GLF and invertase. Thus, a large amount of plasmids containing these genes were produced for future experiments.

Enzymology

Assay for ethanol detection. Simple one-step assay takes ethanol and NAD+ into acetylaldahyde and NADH with use of alcohol Deh (ADH) enzyme. Final NADH concentration can be determined using its molar extinction coefficient and absorbance at 340.0 nm, and is proportionate to initial ethanol concetration. Ethanol concentrations ranging from 0.05mM-0.25mM were assayed with 173U/mL ADH. and absorbances were measured at 340.0 nm.

Results: Final NADH concentrations were calculated to be off by a factor of ten due to unexpectedly low absorbancies. Assay was repeated with increased ethanol concentrations (0.5-10mM), but absorbancies remained lower than expected.

Discussion: It was determined that this assay was now sensitive enough for the range of ethanol concentrations that will need to be detected in our project, therefore we will use other means of ethanol detection using simple primary alcohol detection methods using oxidizing reagents.

Media

Tested growth of NEB 10-ß E. coli cells in BG-11 supplemented with 50 mM glucose. Growth was measured relative to LB broth. Cells were cultured at 37° C with 300 rpm shaking. E. coli showed impaired ability to grow in BG-11 media.

Week 3 - June 13th-19th

E. Coli

Pyruvate Decarboxylase & Alcohol Dehydrogenase

JC: Received pyruvate decarboxylase and alcohol dehydrogenase (PDC/ADH) genes (3054bp) on 6/14/11. Genes were in pUC57 plasmid (AmpR, 2710bp). Transformed PDC/ADH/pUC57 into NEB10 β cells and selected single colonies from LB-Ampicillin plates. Grew liquid cultures in LB-Amp and performed minipreps and nanodrop analysis.

To prepare PDC/ADH for insertion into pSB1C3, 0.5ug of DNA was digested with EcoRI and PstI. Digestions were run on a 0.7% agarose gel, and bands obtained confirmed PDC/ADH (3054bp) and pUC57 (2710bp).

Performed QIAquick PCR Purification of PDC/ADH- EcoRI/PstI digest and pSB1C3- EcoRI/PstI digest using 0.5ug and 0.25ug of DNA respectively. Ligated PDC/ADH and pSB1C3 and transformed into NEB10 β cells. No cells grew on LB-Chloramphenicol plates =(

Bay Laurel Thioesterase

MT: A restriction digest was done on BTE and pSB1C3 with EcoRI and PstI. The digest were checked on a 0.7% agarose gel. The gel confirmed full digestion with EcoRI and PstI on both the BTE and pSB1C3. Overnight ligation was done on the digest at 4C. NEB Beta cells where used for the transformation. Selection was done with chloramphenicol plates and cross selection was done with ampicillin plates.


Cyano

6/13/11: Prepared M-9 Media

Enzymology

This week we ran the Hexokinase glucose/fructose test assay. Prep for the assay consisted of making glucose and NAD+ stock solutions. ASSAY WAS NOT TESTED WITH PGI! The PGI enzyme, responsible for converting fructose into glucose, will be ordered and stored until further testing. Test assay worked perfectly with positive, ideal results. Spectrophotometric analysis reveals NADH concentrations in solution to correlate with original known glucose concentrations, confirming that everything “is working.”

Media

Growth of NEB 10-ß cells was tested in minimal (M9) media supplemented with 36 mM glucose and BG-11 supplemented with 50 mM glucose or 1 mg/mL (18.7 mM) NH₄Cl and 50 mM glucose. The results of this experiment showed negligible growth in all media aside from LB. It was thought that this was likely due to one or more auxotrophies in 10-ß cells.

New England Biolabs technical assistance was contacted, and it was confirmed that 10-ß cells are auxotrophic for leucine. The next experiment will be to see if leucine additions to the media will allow for sufficient growth.

Week 4 - June 20th-26th

E. Coli

Pyruvate Decarboxylase & Alcohol Dehydrogenase

JC: Re-attempted transformation of PDC/ADH/pSB1C3 into NEB10 β cells. Selected single colonies from LB-Chloramphenicol plates and single colony streaked onto LB-Chl and LB-Amp plates to perform plasmid check. Grew liquid cultures in LB-Chl and performed minipreps and nanodrop analysis.

To confirm PDC/ADH insertion into pSB1C3, 0.5ug of PDC/ADH/pSB1C3 was digested with EcoRI and PstI. Digestions were run on a 1% agarose gel, and bands obtained confirmed PDC/ADH (3054bp) and pSB1C3 (2070bp).

PDC/ADH/pSB1C3 was sent to Nevada Genomics Center for sequencing on 6/23/11.

Bay Laurel Thioesterase

MT: 3-2 and 3-3 were the only cell lines that did not grow on the cross selection amp plates. 3-2 and 3-3 were cultured in LBCm34. The cultures were minipreped, nanodroped, and digested with EcoRI and PstI. The digestion was checked with a 0.7% agarose gel. The gel showed that there was full digestion of 3-2 with a band at 2000 (pSBiC3) and 1200 (BTE) which was to be expected, and partial digestion of 3-3. Sample 3-2 was chosen to be sent to the Nevada Genomic Center for sequencing, because it was fully digested with EcoRI and PstI. Sequencing from the Nevada Genomic center proved the successful ligation of Bay Laurel Thioesterase with RBS and Terminator into pSB1C3, and is ready for submission to iGEM.

Cyano
Procedure: GLF and INV were ligated into the iGEM vector PSB1C3. Promoters and integration vector for Synechocystis from iGEM Utah State received. Promoter (petBD, integration vector, GLF, and INV in pSB1C3 were transformed in DH5α cells. Cultures streaked and grown overnight. Colonies selected from streaked plates, liquid cultures grown overnight. Liquid cultures miniprepped to isolate plasmids. Digestion using EcoR1 and PstI performed on each of isolated plasmids including INV and GLF, gels run and imaged. LB with 30% glycerol stock prepared for freezing of E. Coli.

Results/Discussion: The EcoRI and PstI digests did not confirm the successful transformation and isolation of pSB1C3 containing petBD, integration vector, GLF, or INV. Either the digestion was incorrectly performed, or the transformations were unsuccessful. The isolated plasmid DNA needs to be submitted for sequencing to confirm that all genes necessary for PCR and gibson assembly are correctly ligated in the pSB1C3 vector.

Procedure: An apparatus for growing Synechocystis was prepared using a flask and glass tubing connected to an aquarium pump to aerate the culture. Synechocystis was grown in a photobioreactor for four days, and the OD730 was measured once a day to determine growth. Synechocystis was grown in BG-11 medium with either 0% glucose or 50 mM glucose.

Results/Discussion: Over four days, the OD730 did not change for either type of culture. This indicates that the Synechocystis did not grow, and that modifications to the growth apparatus are necessary. A larger volume of culture is necessary, as well as a humidifying device.
6:22/11: Mini prep of glf promotor and DNA digestion

Enzymology
The week consisted of prep work for our first ethanol test assay. We are using an assay method traditionally used for testing for alcohol dehydrogenase (ADH) activity, but can still be used to quantitate ethanol present. Prep work consisted of preparing an ADH stock solution and making buffers, specifically a pyrophosphate buffer which will be used as the assay buffer, and two separate phosphate buffers, one containing Bovine Serum Albumin, used to store and dilute the ADH enzyme. Also prepped were ethanol standards of known concentration, which will be used to “test” the assay next week. Standard were prepared to be with the ranges of expected ethanol secretion.

Media
A time course experiment was performed to test the growth of 10-ß cells in BG-11 supplemented with leucine. Growth was tested in BG-11, BG-11 with NH₄Cl, BG-11 with leucine, and BG-11 with NH₄Cl and leucine. In all cases the media was also supplemented with 50 mM glucose. The results indicated that leucine made no difference in the carrying capacity of our media, and NH₄Cl had a marginal benefit.

Next, testing will be done with different cell lines with the intent of finding a strain that can be readily cultured in BG-11 with minimal supplements.