Team:CSU Fort Collins/Notebook

COLORADO STATE
UNIVERSITY iGEM

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March:

3/12/15:

Made overnight cultures of lac promoter in pSB1C3 and of KillerRed and trans-zeatin in backbones from synthesis.

3/13/15:

Prepared minipreps from overnight cultures of lac promoter, KillerRed, and trans-zeatin plasmids.

3/14/15:

Completed digestion, ligation, and transformation of E. coli with lac:KillerRed and lac:trans-zeatin.

3/17/15:

Checked colonies with colony PCR protocol. Used annealing temperature of 65℃ and extension time of 2 minutes. Ran gel of results. lac:KillerRed colonies 4, 9 were successful.

From left to right: Ladder 1, KillerRed colonies 1-9, ladder 2, trans-zeatin colonies 10-16, positive control

3/18/15:

Miniprepped lac:KillerRed colonies from overnight cultures using kit instructions. Nanodropped results. Re-tried construction of lac:trans-zeatin.

3/19/15:

Checked colonies of lac:trans-zeatin. Results successful.

May:

5/15/15:

Miniprepped overnight cultures of KillerRed in pSB1C3. Attempted to create RBS:KillerRed using SLIM procedure. Made new LB, LB + Amp¹⁰⁰ plates, and LB + Kn⁵⁰ plates.

5/18/15:

Checked May 15th results for RBS:KillerRed on gel; results unsuccessful. Retried procedure using a different thermocycler.

June:

6/4/15:

Miniprepped RBS and P_lac.

6/5/15:

Used PCR to amplify FadD. Annealed at 48.5 ℃ , 55 ℃ , and 62.5 ℃ . Extension for 2 minutes.

6/6/15:

Used PCR to amplify FadD. Annealed at 48.5 ℃ , 55 ℃ , and 62.5 ℃ . Extension for 2 minutes.

6/9/15:

Ran gel on PCR product from FadD

Lane 1: 1 kb ladder Lane 2 & 3: FadD

6/11/15:

Digested Biobrick backbone pSB1C3 (cut sites Spe, Pst), FadD (cut sites Xba, Pst), and P_lac (cut sites Spe, Pst). Ligated FadD+P_lac and FadD+pSB1C3 at 16 ℃ overnight.

6/12/15:

Miniprepped P_trp and Ribosome binding site (RBS) + KR and set up for sequencing. Transformed P_lac + FadD and FadD into pSB1C3

6/14/15:

Made overnight cultures of (RBS+KR)

6/15/15:

Prepare media and overnight cultures for trans-zeatin growth experiment. Ran PCR check on P_lac + FadD and FadD.

Gel 1: Lanes 1 & 2 are FadD in pSB1c3 Gel 2: Lanes 6 & 7 are P_lac +FadD

6/16/15:

Followed procedure for trans-zeatin growth experiment up to a final growth time of 1 day.

6/17/15:

Miniprepped the two successful FadD + pSB1C3 colonies and the two successful P_lac + FadD colonies.

6/18/15:

Ran HPLC procedure for analysis of fermentative products. Miniprepped P_trp again and re-digested and ligated. RBS+KR came back from sequencing correct so this was ligated with P_trp

InterLab: Reconstitute BioBrick parts BBa_J23106: 2015 Kit Plate 1, Well 22A. Size: 35 bp Cm^R and BBa_I13504: 2015 Kit Plate 4, Well 21J. Size: 875 bp Amp^RTransform J23106 and BBa_I13504.

6/19/15:

Transformed P_trp+RBS+KR

6/22/15:

Taq checked P_trp+RBS+KR

InterLab: Reconstitute BioBrick parts BBa_J23101: 2015 Kit Plate 1, Well 20K. Size: 35 bp. Cm^RTransform J23101 (Two plates: unconcentrated and concentrated) . Performed colony PCR on BBa_J23106 and BBa_I13504 transformed colinies.

6/23/15:

Ran gel on Taq check from June 22nd and got no visible results. Received sequencing results for FadD in pSB1C3 and P_lac+FadD and overnight cultures were made to glycerol stock these products.

InterLab: Annotate and restreak. Pick and annotate 16 colonies onto new LB+Cm²⁵ plates. Leave at 37°C overnight. Made overnight cultures of BBa_J23106 and BBa_I13504 transformed colinie

6/24/15:

Made glycerol stocks of FadD in pSB1C3 and P_lac+FadD Attempted to dephosphorylate P_trp backbone but was unsuccessful

InterLab: Colony PCR (16 rxns total). Run PCR products in 2% agarose gel.

The expected size of J23101 sequenced is 318bp, so all colonies look correct. We will move forward using colonies A and F because their bands are clear. Minipreped BBa_J23106 and BBa_I13504 overnights and sent them off for sequencing. Digested J23106 and I13504.

6/25/15:

InterLab: Make overnight cultures of J23101 colonies A and F for Miniprep. Use 2mL of LB + 2uL of Cm (25mg/mL). Leave at 37°C overnight.

6/26/15:

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InterLab: Miniprep J23101 A and F using Promega kit and sent them off for sequencing.Decided to move ahead because the sizes from the gel looked correct. Digested 1ug of J23101 F. Decided to start more overnights from A and F J23101 colonies in case digestion doesn’t work. Sequencing confirmed for J23106 and I13504, digested J23106 and I13504 was done incorrectly because no PCR clean up was used.

6/29/15:

InterLab: Miniprep J23101 A and F. J23106 and I13504 overnight cultures made for glycerol stocking and Miniprep.

6/30/15:

InterLab: Overnight culture of J23101 F for glycerol stock. Minipreped, digested, and overnight ligation performed on J23106 and I13504.

July:

7/1/15:

InterLab:Digest I13504 with XbaI and PstI.Glycerol stock J23101 F. Transformed J23106 and 13504 and grew on Amp²⁵ plates.

7/2/15:

InterLab: Ligation: J23101 and I13504. J23106 and 13504 had no growth. Religated J23106 into 13504

7/3/15:

InterLab: Transformation of ligation reaction didn’t work. Transformed J23106 into 13504 and grew on Amp²⁵ plates.

7/6/15:

Made overnight cultures of P_lac and P_lac+KR in LB+Chloramphenicol (CM)

InterLab: Redo J23101 and I13504 digestions, ligation and transformation. Performed colony PCR on J23106 and I13504 colonies.

7/7/15:

InterLab: Ran J23106 and I13504 colonies PCR results on 1% gel.(pic) colonies from well 3 and 4 showed improper ligation.

7/8/15:

Performed Quick and Dirty experiment and got a lawn of growth so protocol was rethought.

InterLab: Ligation transformation didn’t work. Redid digestions.Performed colony PCR on 14 new colonies from J23106 and I13504 transformation.

7/9/15:

InterLab: Redo J23101 and I13504 ligation and transformation.Ran J23106 and I13504 colonies PCR results on 1% gel.(pic) gels show that transformation was unsuccessful.

7/10/15:

InterLab: Transformation plates had lots of colonies. Annotate and restreak colonies Pick and annotate onto new LB+Cm²⁵ plates. Leave in incubator at 37°C overnight.Re-digest J23106 and I13504 and use heat inactivation instead of PCR clean up kit.

7/13/15:

Performed Quick and Dirty experiment (see google drive) and got a lawn of growth so protocol was rethought.

InterLab: Run PCR products in agarose gel.(Image Interlab) (Image Interlab) Colony #2 looks like the (only) ligated product. J23117+I13504 is about 1.2kb.Ligated J23106 into I13504 and transformed. Grew transformed cells on Amp²⁵ plates.

7/14/15:

Performed Killer Red Experiment (see drive) and KR was expressed.

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InterLab: Make 2 overnight cultures of J23101+I13504 Colony 2. 2mL LB + 2uL Cm (25mg/mL). Ran colony PCR on 14 J23106 and I13504 transformed colonies.

7/15/15:

Killer Red Experiment did not work as planned.

InterLab: Miniprep both cultures. Send one for sequencing with VF2 and VR. Ran J23106 andI13504 PCR products on 1% gel.(pic)Successful

7/15/15:

InterLab: Made J23106 and I13504 overnight cultures for sequencing.

7/17/15:

Created media and prepared flasks for trans-zeatin growth experiment. The experiment needed to be repeated because we suspected that 1 day was not long enough for the trans-zeatin to be created in detectable amounts.

InterLab: Minipreped J23106 and I13504 and sent the off for sequencing.

7/19/15:

Created overnight cultures of strains for trans-zeatin growth experiment.

7/20/15:

Day 1 of trans-zeatin growth experiment.

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InterLab: Although the prefix and J23101sequences are correct, I13504 and the suffix are unclear. The chromatogram signals for these last two regions were also very weak. I think the sequence is correct because Construct 2 group (Dylan, Dakota, Sam and Aidan) sequenced I13504. I will send for sequencing again.

7/21/15:

Day 2 of trans-zeatin growth experiment.

7/22/15:

Day 3 of trans-zeatin growth experiment. Collected final samples of liquid culture. Cells were accidentally thrown away, so the experiment needed to be repeated.

7/28/15:

Started overnight culture of P_lac+KR for Miniprep.

7/29/15:

Anthony Miniprepped P_lac+KR

7/30/15:

Attempted transform, but used wrong competent cells

7/31/15:

Did P_lac+KR transformation with cells with strong LacI promoter

August:

8/1/15:

Plate was too overgrown to do Taq polymerase check so transformation will be redone.

8/3/15:

Transformation on P_lac+KR

8/4/15:

Performed Taq check using Promega “GO Taq”

8/5/15:

Ran gel on PCR and results came back inconclusive as even ladder did not appear under UV light.

8/11/15:

InterLab: Send J23101+I13504 Colony 2 for sequencing again using VR primer.

8/17/15:

Overnight cultures of P_lac and P_lac+FadD were started.

InterLab: If the suffix sequence is correct, I think the I13504 sequence is most likely correct as well. At the mismatched sequences, there was a high background signal. I think this part is good for use in the Interlab measurements.

8/18/15:

ODs were taken on overnight samples of P_lac and P_lac+FadD. Results were inconclusive as P_lac cells grew to a higher OD and much faster than the P_lac+FadD cells. KR+P_lac was transformed and an overnight culture was started. Foil was used the whole time to prevent cells from being exposed to light. Trans-Zeatin+FadD was transformed and an overnight culture was started.

8/19/15:

Made media, autoclaved flasks, and prepared overnight cultures to run trans-zeatin growth experiment. Both plates from August 18th had an acceptable number of colonies and the KR+P_lac colonies were not red.

8/20/15:

Day 1 of trans-zeatin growth experiment.

8/21/15:

Day 2 of trans-zeatin growth experiment.

8/22/15:

Day 3 of trans-zeatin growth experiment.

8/25/15:

Ran PCR with F’i^2 cells and primers for the FadL gene using Phusion DNA polymerase. Reaction was ran in duplicate (2 total)

8/26/15:

Gel was run and results were inconclusive (smear for product 1 and mark around 500bp for product 2) SIx overnight KR cultures were started which contained 5ml LB, 5ul CM, and 2.5ul IPTG. 3 of these were inoculated with colonies of KR+P_lac from 8-8-15. The other 3 were inoculated with P_lac cells from glycerol stocks.

8/27/15:

Ran PCR on FadL. Gel results came back with band of the right size indicating presence of FadL. Started overnight of Lipase L1 from Bacillus Stearothermophilusfor sequencing and an overnight of both P_lac and P_lac+FadD.

September:

9/7/15:

Made media and prepared bioreactors for trans-zeatin prototype (bioreactor experiment).

9/8/15:

Transformed P_lac+Lipase L1, P_lac+FadL, P_lac+FadD+FadL, and P_lac+KR cultures.

9/9/15:

PCR check done for all four transformations in replicates of 8. Taq DNA pol was used and the “Go Taq” DNA polymerase protocol from Promega was used.

9/10/15:

Made 50 mL overnight cultures for bioreactor experiment.

9/11/15:

Day 1 of bioreactor experiment.Gels were run on products 1-7 of P_lac+Lipase L1, P_lac_FadD+FadL, and P_lac + FadL.

9/12/15:

Day 2 of bioreactor experiment.

9/13/15:

Day 3 of bioreactor experiment. Took samples and extracted zeatin from bioreactor and previous experimental results using single-phase extraction procedure.