iGEM 2017 — Team Lund

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# Team:Lund/Notebook

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# Notebook

## June

Week 26 - 2017/06/28 - 2017/06/30

* Obtaining the pETDuet-1 plasmid backbone
* Cell cultivation (*E. coli* BL21-DE3) with pETDuet-1 plasmid
* Plasmid isolation (pETDuet-1) using GeneJET Plasmid Miniprep Kit
* Spectrophotometric analysis of plasmids
* Too low plasmid concentration, therefore we ordered commercial plasmid backbones

## July

Week 27 (2017/07/03 - 2017/07/07)

* Cell cultivation (*E. coli* BL21 and *E. coli* TG1)
* Preparation of competent cells using the Inoue Method for both *E. coli* strains
* Freezing of competent cells

Week 28 (2017/07/10 - 2017/07/14)

##### InterLab attempt 1

* Transformation of competent (*E. coli* DH5ɑ) cells
* The transformation did not succeed
* Competent cell test (*E. coli* TG1 and BL21) using Competent Cell Test Kit
* Successful competent TG1 and BL21 *E. coli* cells
* Have not calculated the cell efficiency, as cell competency test was only a test experiment

Week 29 (2017/07/17 - 2017/07/21)

##### InterLab attempt 2

* Plating of competent cells (*E. coli* DH5ɑ)
* Competent cells grew poorly, therefore non-competent ones were plated as well

Week 30 (2017/07/24 - 2017/07/28)

* Competent cell test (*E. coli* DH5ɑ)
* Preparing competent cells from the non-competent batch of *E. coli* DH5ɑ cells
* Low effectiveness of the competent cells was measured
* The commercial pETDUet-1 plasmids have arrived

## August

Week 31 (2017/07/31 - 2017/08/04)

* The pETDuet-1 plasmids were sent for gene synthesis
* Transformation of competent *E. coli* DH5ɑ cells
* The transformation did not succeed again, we suspect the cells were in a bad condition
* Asked iGEM team from Gothenburg, to get some *E. coli* DH5ɑ cells

Week 32 (2017/08/07 - 2017/08/11)

* The second batch of pETDuet-1 plasmids arrived
* Received DH5ɑ cells from iGEM team Chalmers Gothenburg
* Making DH5ɑ competent cells

Week 33 (2017/08/14 - 2017/08/18)

* pETDuet-1 plasmids sent to Eurofins
* Competent cell test (*E. coli* DH5ɑ, BL21, NEB5ɑ)
* Transformation of competent *E. coli* BL21 cells with test BioBricks
* The transformed cells for the InterLab study did not grow
* Contacted iGEM Headquarters to send us more plasmid DNA
* Contacted iGEM DTU Denmark to send us already transformed DH5ɑ cells for the InterLab study

Week 34 (2017/08/21 - 2017/08/25)

* Plasmid isolation from transformed *E. coli* BL21 cells with test BioBricks
* Restriction digestion
* Agarose gel electrophoresis

## September

Week 35 (2017/08/28 - 2017/09/03)

* Gel extraction of test BioBricks
* Digestion of the plasmid backbone and ligation of devices 1G1 and 1G2
* Agarose gel electrophoresis
* Transformation, which resulted in red (containing RFP) and white cells (should not be there)
* Inoculation  of the media with both red and white cells growing after transformation to get more DNA to send for sequencing
* Plasmid isolation and freezing of DNA
* Repeating InterLab with cells from iGEM DTU Denmark
* InterLab measurements resulted in negative values
* Repeated the plating of the cells by concentrating already grown bacterial culture
* After 26 h incubation no cells grew

Week 37 (2017/09/11 - 2017/09/17)

* Digestion and ligation of gBlocks GFP1-9 and gBlock GFP1-9 device
* Agarose gel electrophoresis to see how successful the ligation was
* Transformation of the ligated gBlocks to *E. coli* BL21
* Transformation of *E. coli* DH5ɑ cells with the new batch of devices
* Plating of the transformed cells
* Preparing 10 mL culture media

Week 38 (2017/09/18 - 2017/09/25)

* Inoculation of 100 mL media with the overnight culture
* InterLab measurements
* Digestion and ligation of the GFP1-9 device and GFP1-9; ligation was done overnight
* Digestion and ligation of devices for interlab; ligation was done overnight
* Colony PCR of InterLab devices to check if the colonies appearing white on the media had GFP inserted
* PCR products were run on agarose gel

## October

Week 39 (2017/09/25 - 2017/10/01)

* InterLab measurements
* Preparing glycerol stocks of *E. coli* BL21 cells with inserted GFP1-9 and GFP1-9 device
* Plating of cells from the glycerol stocks to check if they were prepared correctly
* Digestion and ligation of the NahR BioBrick
* Transformation of NahR BioBrick to *E. coli* BL21
* Colony PCR of the transformed NahR BioBrick
* Plasmid purification of GFP1-9 and GFP1-9 device BioBricks

Week 40 (2017/10/02 - 2017/10/08)

* Harvesting the cells cultivated from the glycerol stock
* Sonication of cells with the GFP1-9 device insert
* Plating cells with GFP1-9 device insert to measure any possible background fluorescence
* Running SDS-PAGE to check for expression of GFP1-9
* Digestion and ligation of NahR-GFP1-9 device and NahR-sfGFP device
* Transformation of ligated NahR-GFP1-9 device and NahR-sfGFP device
* Transformation of NahR in pETDuet vector to *E. coli* BL21, plating of the cells - no growth
* Agarose gel electrophoresis to check if the ligation succeeded - it did not
* Repeated digestion and ligation of NahR-GFP1-9 device and NahR-sfGFP device
* Repeated transformation of ligated NahR-GFP1-9 device and NahR-sfGFP device

Week 41 (2017/10/09 - 2017/10/15)

* Colony PCR of the plated GFP1-9 device
* Plating of competent *E. coli* BL21 cells and *E. coli* BL21 cells with GFP1-9 insert from the glycerol stock
* Digestion and ligation of ER-ɑ device, NahR-GFP1-9  device and NahR-sfGFP device
* Agarose gel electrophoresis of digested ER-ɑ device, NahR-GFP1-9  device and NahR-sfGFP device products
* Transformation of chromoprotein BioBrick (Bba\_K1033910) using *E. coli* BL21 and TG1 strains - lawn of bacteria on plates
* Colony PCR of plated cells with chromoprotein insert - no insert could be multiplied
* Transformation of ligated devices ER-ɑ, NahR-GFP1-9, NahR-sfGFP, both plated and also inoculated to 10 mL LB media
* No growth of the three transformed devices on a plate, very low OD as well
* Digestion/ligation of ER-ɑ

Week 42 (2017/10/16 - 2017/10/22)

* Transformation of ER-ɑ to *E. coli* BL21, GFP1-9 and sfGFP (already in a pETDuet-1 plasmid) to both *E. coli* BL21 and TG1 - no growth (ER-ɑ) or lawn of bacteria (inserts in a pETDuet-1 plasmid)
* Digestion of GFP1-9 device, ER-ɑ part and ER-ɑ device
* Agarose gel electrophoresis - no bands for GFP1-9
* Repeated transformation of chromoprotein BioBrick to *E. coli* DH5-ɑ, BL21 and TG1 strains - no growth
* Replating of transformed devices in pETDuet-1 plasmid (ER-ɑ, GFP1-9 and sfGFP) - growth on all plates
* Replated cell colonies were inoculated to 5 mL LB media over night
* Digestion of GFP1-9 for agarose gel - smeary bands
* Repeated transformation of GFP1-9 device, ER-ɑ part and ER-ɑ device in a pETDuet-1 to *E. coli* DH5ɑ using a standard and quick transformation protocol
* Results: standard protocol - many colonies, quick protocol - lawn of bacteria
* Digestion and ligation of GFP1-9 part and device, ER-ɑ part and device
* Inoculation of cells containing GFP1-9 device, sfGFP and B21 competent cells to 5 mL LB media
* Freezing and thawing of the overnight culture
* SDS-PAGE to check for the expression of the proteins

Week 43 (2017/10/23 - 2017/10/29)

* Transformation of the samples being ligated overnight using quick and standard protocol
* Colony PCR to check wether ligation succeeded
* Agarose gel electrophoresis for checking if the inserts are of correct length
* Noticed excessive growth on plates with ampicillin, where there should not be any growth - inoculation of LB media with cells containing different devices, by using both antibiotics, to see where the growth will appear
* Inoculation of LB media containing ampicillin and chloramphenicol with competent cells to see if the antibiotics were the problem
* Purification of plasmids carrying GFP1-9
* Digestion of the latter plasmids with one and two restriction enzymes
* Agarose gel electrophoresis to check the result of the digestion
* Repeated digestion for much longer time
* Colony PCR of purified plasmids
* Agarose gel electrophoresis for checking the presence of inserts

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##### Home

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##### Team

* [Team](/Team%3ALund/Team)
* [Collaborations](/Team%3ALund/Collaborations)
* [Attributions](/Team%3ALund/Attributions)

##### Project

* [Description](/Team%3ALund/Description)
* [Design](/Team%3ALund/Design)
* [Results](/Team%3ALund/Results)
* [InterLab](/Team%3ALund/InterLab)
* [Parts](/Team%3ALund/Parts)
* [Protocol](/Team%3ALund/Experiments)
* [Notebook](/Team%3ALund/Notebook)

##### Model

* [Results](/Team%3ALund/Model)
* [Model derivation](/Team%3ALund/Modeling/ModelDerivation)
* [Parameter estimation](/Team%3ALund/Modeling/ParameterEstimation)
* [Implementation](/Team%3ALund/Modeling/Implementation)

##### Safety

* [Safety](/Team%3ALund/Safety)

##### Practices

* [Overview](/Team%3ALund/HP/Silver)* [Integrated](/Team%3ALund/HP/Gold_Integrated)* [Engagement](/Team%3ALund/Engagement)* [Media](/Team%3ALund/HP/Media)* [Communication](/Team%3ALund/Communication)

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223 62 Lund
Sweden
igemlund@gmail.com

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