iGEM 2016 — Team WashU_StLouis

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# Team:WashU StLouis/Notebook

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# Notebook

## Reading about labwork is *almost* as fun as doing labwork

| June | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  | 1 | 2 | 3 | 4 |
| 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| 12 | 13 | 14 | 15 | 16 | 17 | 18 |
| 19 | 20 | 21 | 22 | 23 | 24 | 25 |
| 26 | 27 | 28 | 29 | 30 |  |  |
|  |  |  |  |  |  |  |

| July | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  | 1 | 2 |
| 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| 10 | 11 | 12 | 13 | 14 | 15 | 16 |
| 17 | 18 | 19 | 20 | 21 | 22 | 23 |
| 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| 31 |  |  |  |  |  |  |

| August | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  | 1 | 2 | 3 | 4 | 5 | 6 |
| 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| 28 | 29 | 30 | 31 |  |  |  |
|  |  |  |  |  |  |  |

| September | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  | 1 | 2 | 3 |
| 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 11 | 12 | 13 | 14 | 15 | 16 | 17 |
| 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| 25 | 26 | 27 | 28 | 29 | 30 |  |
|  |  |  |  |  |  |  |

| October | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  | 1 |
| 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| 9 | 10 | 11 | 12 | 13 | 14 | 15 |
| 16 | 17 | 18 | 19 | 20 | 21 | 22 |
| 23 | 24 | 25 | 26 | 27 | 28 | 29 |
| 30 | 31 |  |  |  |  |  |

❮❮
❮
❯❯
❯

No lab work done

No lab work done

* First day in lab!
* Made 1L of LB Media

No lab work done

No lab work done

No lab work done

* Performed gDNA Extraction on E. Coli MG1655 and Synechocystis 6803

* Designed PCR primers for *pgk*, *pck*, *fldA*, *petF*, ATP Synthase 1, and ATP Synthase 2

No lab work done

* Primers Arrived
* Ran PCR on ATP Synthase 2 , **pgk**, **fldA**, **petF**, and pdCas9 plasmid
* Ran all PCR products on gel and recovered ATP Synthase 2, **fldA**, and **petF**
* Ran gradient PCR on pdCas9 plasmid

No lab work done

No lab work done

* Made and ran gel for pdCas9
* Ran PCR on ATP Synthase 1, **pgk**, and **pck**
* Performed gel extraction on yesterday’s ATP Synthase 2
* Ran **pck**, **pgk**, and yesterday’s gradient pdCas9 PCR products on gel and recovered all genes
* Performed gel extraction on pdCas9, *fldA*, *petF*
* Ran PCR on psc101 and psc102 plasmid
* Ran psc101 and psc102 PCR products on gel - were recovered
* Ran gradient PCR on ATP Synthase 1
* Made agar plates (forgot the antibiotic)
* Ran golden gate one pot assembly for *fldA* *petF* genes and the pdCas9 plasmid

* Ran ATP Synthase 1 PCR products on gel - no bands seen
* Gel Extraction for *pgk*, *pck*, psc101, and psc102 plasmid
* Ran PCR on ATP Synthase 1 (3rd times the charm)
* Ran PCR products on gel - WE SAW BANDS! ATP SYNTHASE 1 RECOVERED
* Reran PCR on *pgk*, *pck*, psc101, and psc102 plasmid because we did not get high enough concentration during gel Extraction
* Made new agar plates (with the chloramphenicol)
* Transformed the *petF*, *fldA* plasmids into competent cells
* Plated colonies on agar plates and set in incubator

* Checked for colonies (both genes were successful)
* Picked colonies and incubated overnight
* Ran yesterday’s PCR products on gel - bands were light but all were recovered
* Gel Extraction for *pgk*, *pck*, psc101, and psc102 plasmid
* Reran PCR on *pgk*, *pck*, psc101, and psc102 plasmid because we did not get high enough concentration during gel extraction
* Ran PCR products on gel - all were recovered
* Gel Extraction for *pgk*, *pck*, psc101, and psc102 plasmid

* Miniprepped plasmids for *fldA*, *petF*
* Ran sequence PCR on both plasmids
* Ran PCR product on gel to confirm
* Clean and Concentrated PCR product
* Reran PCR on *pgk*, *pck*, psc101, and psc102 plasmid because we did not get high enough concentration during gel extraction (for the fourth time)

* Prepared DNA for sequencing
* Delivered DNA to WashU Med School
* Added DPnI to psc101 and psc102 gel extract products, incubated for an hour, did Clean and Concentrate, measured conc
* Ran Golden Gate assembly on *pck*, *pgk*, and ATP Synthase

* Electroporation with golden gate products
* Redid golden gate for *pck*, *pgk*, ATP synthase (did not run properly first time)

* Electroporation with better golden gate products
* Picked colonies for worse golden gate for *pck*, *pgk*, ATP synthase

* Miniprep worse ATP, *pck*, *pgk* golden gate
* Seq PCR worse ATP, *pck*, *pgk* golden gate
* Picked colonies for better golden gate for *pck*, *pgk*, ATP synthase

* Made frozen stock of better golden gate *pck*, *pgk*, ATP synthase
* Miniprepped better golden gate *pck*, *pgk*, ATP synthase (low concentration)

* Ran sequencing PCR for *pgk*, *pck* (4 colonies each)
* Gel Extraction for *pgk*, *pck* PCR product (no bands)

* Ran sequencing PCR for *pck* and *pgk* on a gradient
* Gel Extraction for *pck* PCR product was successful, not *pgk*

* Ran sequencing PCR for *pgk* on a different gradient
* Gel Extraction for *pgk* PCR product was not successful

No lab work done

* Picked four new colonies of *pgk*
* Started 4 new cultures from frozen stock

* Made frozen stock of the 4 new colonies
* Miniprepped all 8 *pgk* cultures and measured concentrations
* Ran gradient PCR on all 8 *pgk* plasmids
* Prepped and sent ATP synthase and *pck* in for sequencing

No lab work done

* Mixed new *pgk* sequencing primers

* Ran gradient PCR on all 8 *pgk* plasmids

No lab work done

No lab work done

No lab work done

4th of July!

No lab work done

* Sequencing results for ATP and *pck* were not good
* Reran seq PCR on ATP Synthase (bad results)
* Picked new ATP colonies

* Miniprepped overnight cultures of ATP 5-8 and forgot to make frozen stock
* Ran PCR on ATP plasmids 5-8
* PCR for *pgk* with new primers with gradient
* Ran *pgk* PCR product on gels
* Ran golden gate on *pgk* PCR gel extract

* Electroporation with golden gate *pgk* product

* Picked 4 *pgk* products and inoculated them overnight

* Made frozen stock of new 4 *pgk* cultures
* Miniprepped cultures
* Seq PCR on new cultures
* All 4 cultures showed correct bands
* Clean and Concentrate *pgk* PCR product
* Prepped *pgk* for sequencing

* Delivered *pgk* for sequencing

* New ATP synthase sequencing primers and *pck* regular primers came in
* Seq PCR of ATP synthase plasmids with new sequencing primers. No bands
* Picked 4 new ATP synthase colonies (again)
* Inoculated MG1655 from frozens stock to make more genomic DNA

* Extracted genomic DNA from MG1655
* Miniprepped new ATP cultures
* Ran sequencing PCR on new ATP and got no bands at all. Will use newly transformed cells tomorrow
* Ran PCR with *pck* primers, got light bands
* Reran PCR with *pck* products

* Ran *pck* on gel
* Gel extracted *pck* and combined gel extracts to improve concentration
* Golden gate ligation on *pck*
* Ran ATP sequencing PCR again with different backbone primers

* Ran gel from yesterday’s ATP PCR. No bands
* Ran PCR for *pfo*
* Incubated *pck* and BioBrick cells

* Miniprepped *pck* and biobrock genes (HSP)

No lab work done

* Ran ATP seq PCR gradient again. No bands. Done with ATP
* BioBrick digestion, ligation, transformation with HSP and GFP
* Ran PCR seq for *pck* and gel extract

* Delivered *pck* sequence to med school
* Ligated backbone for BioBrick
* Extracted more genomic DNA from MG1655
* Gradient PCR on *pfo*
* Received 𝛥*aceE* cells from Yale
* Rehydrated and incubated 𝛥*aceE* cells

* Ran PCR and gel extract for *fldA* backbone and *petF* backbone
* DPn1 the two backbones
* golden gate ligation on *pfo* gene and two backbones
* Streaked colony from 𝛥*aceE* plate onto LB/kan plate

* Making media for competent cell protocol
* Made new LB with MgSO4 for chemical transformation

* Digested BioBrick miniprep to check length

* Chemically competent cell protocol with 𝛥*aceE*
* Miniprepped *pfo*-*petF* and *fldA*-*petF*
* Seq PCR miniprepped *pfo*-*petF* and *fldA*=*petF*
* Remade LB with MgSO4 with correct concentration

No lab work done

* Restarted comp cell protocol (3rd times the charm)
* Clean and Concentrate junction 2 for *pfo*-*fldA*
* Conducted HSP test in LB

* Redid HSP test in minimal media M9
* Recultured BioBrick and DH10B control

* Reset up BioBrick experiment but grew diluted cells with LB instead of M9, so we have to do it again

* Redid HSP test in minimal media M9

No lab work done

No lab work done

No lab work done

* Chemical transformation of *pfo*-*petF*, *pfo*-*fldA*, *petF*, *fldA* into knockout strain, plated 80 and 240 ul, also control
* Part 1 of interlab study (LUDOX)
* Transforming 5 interlab plasmids

* Picked and incubated electron donor colonies plated yesterday
* Picked and incubated interlab colonies plated yesterday (2 per construct)

* Made frozen stock of incubated colonies and interlab parts
* Redid BioBrick experiment at 37C

No lab work done

No lab work done

No lab work done

* Started induction but had to stop bc no frozen stock of *fldA*-*pfo* or *petF*-*fldA* in DH10B
* Transformed *fldA*-*pfo* and *petF*-*pfo* into DH10B

* Made frozen stock of 𝛥*aceE*
* Made 60% ethanol
* Incubated 8 constructs for biotin assay w/ methanol extraction (4 constructs and blank in DH10B and 𝛥*aceE*)

* Methanol extraction assay (grew cells and froze pellets)

* Assayed *petF*-*aceE* cells. Absorbance was too low to be meaningful

* Ran preliminary sonication test
* Incubated large volume of DH10B

* Ran large sonication test on DH10B to decide protocol

* Incubated large volume of DH10B

* More sonication tests

* Made ATP standards from assay instructions

* Made more LB

* Inoculated *pgk*, *pck*, DH10B for assay tomorrow

* ATP luminescence assay
* Inoculated 4 constructs and blank in 𝛥*aceE* cells for tomorrow

* Nothing grew, couldn’t do induction

* Inoculated DH10B constructs and control from frozen stock

* DH10B electron donor induction, froze pellets, measured OD600
* Re-transformed constructs into 𝛥*aceE* cells

* Sonicated and assayed pellets for biotin (1 minute, 40 amp, 1mL PBS)
* Reinoculated 𝛥*aceE* constructs in all antibiotic combinations to determine problem, in LB and plates

* Visited Monsanto

* Measured absorbance data from biotin in DH10B cells
* Did ATP Assay and got luminescence data
* Incubated all four 𝛥*aceE* electron donor constructs from frozen stock plates and original plates and incubated 𝛥*aceE* and DH10B

* Nothing grew from yesterday's inoculations
* Interlab study

* Remade competent cells
* Finished interlab study (FITC) and sent to iGEM HQ

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

* Picked colonies from 4 new 𝛥*aceE* plates and incubated

* 𝛥*aceE* cell induction

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

* Incubated *pck*, *pgk*, DH10B from frozen stock

* Ran ATP assay
* BioBrick digestion and ligation
* Sonicated 𝛥*aceE* pellets
* Ran 𝛥*aceE* cell absorbance scan
* Ran biotin assay on 𝛥*aceE* cells

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

* Transformed ligated *petF* and *pck* BioBrick plasmids into DH10B (wrong antibiotic)

No lab work done

* Transformed ligated *petF* and *pck* BioBrick plasmids into DH10B (kan = right)
* Wrapped *pck* and *pgk* plasmids to be sent to Cardiff University iGEM

* Small colonies grew on all plates
* Picked colonies and inoculated
* Sent plasmids to Cardiff University iGEM

* Miniprep on *pck* and *petF* BioBrick
* Reusupended TetR-pTet gBlocks

* Digestion/Ligation/Transformation of pSB1C3 and tetr-ptet BioBrick

* TetR-pTet colonies grew, picked colonies

* Miniprepped TetR-pTet BioBrick

* digested BioBricks to check bands sizes

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

No lab work done

* Ran PCR on BioBrick plasmids

* Ran PCR products on gel
* Only *petF* showed up

* digestion of *pck*, TetR-pTet, and pSB1C3 with EcoRI

* Digestion of *pck*, TetR-pTet, and pSB1C3 with PstI
* Ligated *pck* and TetR-pTet to pSB1C3
* Transformed into DH10B

* Only TetR-pTet grew
* Ran colony PCR with 8 *pck* colonies and 8 TetR-pTet colonies

* Ran colony PCR products on a gel but got no bands
* Miniprepped pTet1-8
* Ran PCR on pTet 1-8 plasmids to check if insert was present
* Ran PCR product on gel, got the band!
* Prepped *petF*, *pck*, and TetR-pTet to be sent to iGEM HQ

* Last day in lab!
* Sent parts to iGEM!

WIKIFREEZE

Done in lab

Prepping for Jamboree

Prepping for Jamboree

Prepping for Jamboree

Prepping for Jamboree

Prepping for Jamboree

Prepping for Jamboree

Fly to Boston for Jamboree

GIANT JAMBOREE

GIANT JAMBOREE

PRESENTATION

Fly back to St. Louis

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