iGEM 2017 — Team Uppsala

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# Team:Uppsala/Notebook

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* [Safety](https://2017.igem.org/Team%3AUppsala/Safety)

### Week: 07/06–16/06

– A safety demonstration was done by the Lab technicians and Project managers which enabled us to know the fire escapes, first aid and emergency contacts. Lab space was assigned.
– Buffers and stock solutions were prepared. LB media and LB-agar plates with different antibiotics (Kanamycin, Chloramphenicol and Tetracycline) were prepared.
– *E. coli DH5α* competent cells were prepared. Ordered constructs for the three genes (CaCCD2, CsADH2946 and UGTCs2) were digested with available enzyme (E and S) and ligated to Linearized plasmid backbones from the iGEM Kit.

- Prepared competent cells were tested with the iGEM Competent Cells Test kit. No growth was observed. Possible causes: Faulty competent cells, improper digestion or transformation protocol.
- Made DH5α competent cells with another new protocol to be tested and used later.

### Week: 19/06–22/06

– Testing of the competent cells made last week was done with the competent cells test kit. There was growth suggesting that competent cells were working good.
– To test if the restriction digestion, ligation and transformation worked well, they were repeated again with the newly prepared and tested competent cells above. No growth was observed even when high concentrations were plated, suggesting that the fault was in the Restriction digestion, ligation steps

### Week: 26/06–30/06

– On expert suggestion, it was understood that the enzymes need few extra bases on the ends for efficient activity. Thus we designed and ordered primers that would add overhangs to our constructs. Other reason could be that the promoters were too strong and the genes were toxic to the cell. So a set of primers were also designed to remove promoter from between the prefix and RBS.
– To circumvent the high toxicity of the gene, we decided to clone the devices in low copy plasmids. For that low copy plasmids were identified from the master strain database of glycerol stock at the lab, grown overnight in agar plates, grown overnight in LB and plasmids were purified.

- Digestion was tested with X and S to optimize the protocol for digestion

### Week: 03/07–7/07

– The primers designed last week were tested and PCR Parameters optimized. PCR amplicons were electrophoresed and positive sections were eluted from the gel and their concentrations measured.
– Low copy plasmid backbones purified last week were digested and linearized. The PCR amplified constructs were digested and ligated to Low copy plasmid backbone. Transformation was done
– High copy plasmid backbones from the glycerol stock were purified.

### Lambda red integration:

– Planning/research for project.
– Made competent cells of IS150 and gsp strain.

### Week: 10/07–14/07

– Low copy plasmids transformed last week were re-streaked. Colony PCR followed by gel electrophoresis was done on them.
– Positive colonies, screened from colony PCR were re-streaked and glycerol stock was made for them.
– Electroporation-competent DH5α cells were prepared.
– The PCR amplified constructs were restricted digested and ligated to high copy plasmid backbones cells. These plasmids were then transformed by heat-shock.
– More low copy plasmid for each antibiotics with Red fluorescent protein RFP were purified from the master stock.
– UGTCs2 gene was cloned in pSB1C3 plasmid backbone since using tetracyclin was being troublesome.
– Electro-competent cells were tested with cloned genes. No growth was observed. Possible cause: cells died during electroporation. Desalt the ligated DNA.
– Colony PCR of the positive colonies (white colonies) obtained from heat shock transformation of cloned genes in high copy plasmids. No bands visible.

### Lambda red integration:

– Tested transduction with bgl strain as the donor and MG1655 as receiver (*cat-sacB* will be transduced). 1 colony was found on the plate with chloramphenicol. This one was restreaked on chloramphenicol which then had more growth.
– Tried to insert *crtI* gene in IS150 strain and *dhfr* gene in gsp strain using lambda red. The IS150 strain grew on sucrose (indicating that the *sacB* sequence had been replaced) but tetracycline had not been added so the strain could have lost the lambda red plasmid. The gsp strain had strange growth pattern.

### Week: 17/07–21/07

– Fresh start of restriction digestion and ligation of PCR amplified constructs into high copy plasmids. Used both heat shock (with commercial competent cells) and electroporation.
– Positive (white) colonies were restreaked and colony PCR was done. No bands were observed.
– Troubleshooting of colony PCR. Optimised PCR parameters and new buffer components.
– SDS-PAGE parameters were optimised and a test run was done with BSA dilutions. No bands were observed in the first run due to improper buffer. Troubleshoot and Run again!

### Lambda red integration:

– PCR of inserts *crtI*, *dhfr*, *crtZY* and *crtEB*. Gel elecrophoresis for verification (see figure below).
– PCR of inserts *crtZY* and *crtEB*. Gel electrophoresis for verification but there was nothing on gel purification of inserts *crtI* and *dhfr*.
– Gel electrophoresis of pooled PCR products of insert *crtEB* and *crtZY* from 17/7 (see figure below). Electroporation of purified *crtI* gene in IS150 strain and purified *dhfr* gene in gsp strain. Put overnight on shaking water bath.
– The overnight cultures of IS150 strain was streaked on tetracycline and sucrose plates and gsp strain was streaked on tetracycline and trimetoprim plates. Gel electrophoresis of *crtEB* insert (see figure below).

### Week: 24/07–28/07

– Positive colonies carrying construct in tetracycline backbone from last week were re-streaked to check for viability.
– Buffers for immobilised metal affinity chromatography (IMAC) were prepared and degassed. IMAC column was packed.
– Positive cells (white colonies) from containing genes in high copy plasmids were grown overnight and sonicated. The lysates were purified by IMAC.
– More LB agar plates with antibiotics were prepared.

### Lambda red integration:

– PCR of insert crtEB. Restreak clones of IS150 strain from 21/7 on sucrose and tetracycline plates and gsp strain from 21/7 on and tetracycline and trimetoprim plates. Liquid cultures with same respective antibiotics for the strains were made.
– Gel electrophoresis of *crtEB* PCR product from 24/7. Restreak colonies of IS150 strain from 24/7 plates on sucrose plates then chloramphenicol plates. Prepare cultures for transduction of IS150 by growing overnight in P1 LB. Purify *crtZY* and *crtEB* and use nanodrop on all purified inserts to check DNA concentration. Ordered new primers for *crtEB*.

– gsp strain did not grow on plates but did in liquid culture. 5 of 8 colonies of IS150 strain from 25/7 grew on sucrose but not chloramphenicol. New plates were made (tet + chlor, tet + tmp, tet + suc, chlor). Transduction of bgl strain as the donor and IS150 as receiver. Take some gsp liquid culture and put in some tet + sucrose to make sure it's our strain that's growing. Make liquid cultures with gsp (*dhfr*) in tet + tmp with the different tet to check if antibiotics work.
– No growth on transducer IS150 from 26/7. Start overnight culture of bgl strain to make lysate (donor strain). Colony PCR of the colonies from 26/7 that grew on sucrose but not on chlor and gel electrophoresis to check inserts. Make tet + chlor and tet + tmp plates. Start overnight cultures of IS150 strains with inserts that were confirmed by colony PCR.
– Colony PCR of gsp strain and gel electrophoresis

### Week: 31/07–4/08

– To confirm the presence of genes in the colonies, Screening was performed by restriction digestion and electrophoresis.
– Troubleshooting of colony PCR was done with commercial Taq polymerase master mix.
– Biobrick from 2010 Slovenia Team containing 5 enzymes *crtEBIYZ* was transformed to *E. coli* by heat shock.
– Fresh batches of 3 selected positive colonies (one for each enzyme) were grown overnight in TB and lysed for sonication. Lysates were purified by IMAC.
- Eluted fractions were subjected to SDS-PAGE.
- The three colonies with recombinant plasmids were grown and plasmid prep was done. Restriction digestion and gel electrophoresis was done for screening.
- Fresh, presumably positive colonies (white colonies) were selected from master plates and re-streaked. Colony PCR was done for them followed by electrophoresis
- Gel chromatography and SDS was done for IMAC purified elutes.
- A second round of screening by digestion was done with modifications as the plasmids and the construct were of similar size therefore needed better resolution.
- Making all the constructs in pSB1C3 for biobrick submission.

### Lambda red integration:

– Start overnight culture of bgl with chlor for lysate.
– Make lysate for transduction with bgl strain as donor. Start overnight culture of gsp strain (to make competent the next day) and one of the colonies from 27/7 of the IS150 strain that had *crtI* insert (to have as receiver for transduction next day).
– Finish lysate with bgl strain as donor. Make competent cells of gsp strain. Transduction of bgl strain as the donor and IS150 (prepared 1/8) as receiver and streak on tet+chlor plates.
– No colonies on transduced plates from 2/8. Electroporation of dhfr insert in gsp strain.

### Week: 7/08–11/08

### Lambda red integration:

– PCR of *crtEB* insert. Buffer/media preparation. Redo transduction from 2/8 and electroporation from 3/8. Start overnight culture of bgl with chlor for lysate and IS150 strain that had *crtI* insert (to have as receiver for transduction next day).
– Gel electrophoresis of *crtEB* PCR product from 8/8 (see figure 8). Make lysate for transduction with bgl strain as donor (in 30 °C and 37 °C). Transduction of bgl strain (lysate made 2/8) as the donor and IS150 (prepared 8/8). Start overnight culture of the IS150 (to have as receiver for transduction next day).
– No colonies on transduced plates from 9/8. Make plates and SOB. Finish lysate (30 °C and 37 °C) from 9/8. Purify *crtEB* from PCR 9/8. Electroporation of *dhfr* insert in gsp strain and *crtZY* in gsp strain. Transduction of bgl strain (lysate made same day) as the donor and IS150 (prepared 9/8).
– No colonies on transduced plates from 10/8. Streak electroporated gsp strains from 10/8 on tet+tmp+chlor plates for strain with *dhfr* and tet+suc plates for *crtZY*).

### Week: 14/8–18/8

### Lambda red integration:

– Electroporation of *crtEB* insert in IS150 strain and *crtZY* in gsp strain. Start overnight culture of gsp with *crtZY* + *dhfr* and IS150 with *crtI* + *crtEB*.
– Make lysate for transduction with gsp strain as donor. Transduction of bgl strain (lysate made same day) as the donor and IS150 (prepared 14/8).
– No colonies on transduced plates from 15/8.

### Week: 21/8–27/8

### Lambda red integration:

– P1 transduction of bgl::*cat-sacB* into the IS150::*CP25-crtI* strains + wt control + cam control
Incubate 30 minutes in 37 °C
Plate *crtI* recipients on cam+tet, incubate in 30 °C
One control on only cam, 30 °C
Plate wt control on cam, incubate in 30 °C
Run screening PCR of IS150::*crtI* and gsp::*crtYZ* strains
– Gel analyze *crtI* and *crtYZ* screening – all clones correct size!
Purify PCR products *crtI* and *crtYZ*
Check transduction plates – the transduction worked well, more than 100 colonies per plate.

– Run screening PCR of IS150::*crtI* and gsp::*crtYZ* strains
Gel analyze PCR products
*crtI* screening (4 wells) – weak bands
*crtYZ* screening (3 wells) – good bands
Start overnight cultures *crtI* + bgl::*csb*
– Purify PCR products
Send for sequencing
*crtI* – use IS150\_seq\_2, crtI\_scr\_1 and crtI\_scr2 (IS150\_scr\_R)
*crtYZ* – use crtZY\_scr\_1,crtZY\_scr\_2 and gsp\_scr\_R (gsp\_ins\_R)
Freeze *crtI* + bgl::*cat-sacB* strains + streak them on plate and leave in RT over the weekend

### Week: 28/8–3/9

– Ordered more UGTCs2 from IDT
– linearised iGEM plasmid pSB1C3-J04500 using Phusion PCR and transformed CsADH2946 construct into the plasmid using Gibson assembly.
– Transformed into commercial competent TOP10 *E. coli* cells.

![](https://static.igem.org/mediawiki/2017/a/aa/T--Uppsala--notebook_gel1.png)

### Lambda red integration:

– Sequencing results showed that the clones are correct.
Streak up from -80 °C
Eco gsp::*crtZY* /pSIM5-tet clone 1

– Start overnight cultures of *crtI* and *crtZY* strains (Salt free LB + tet, 30 °C)
Eco gsp::crtZY /pSIM5-tet clone 1
Eco IS150::*crtI* bgl::*cat-sacB* /pSIM5-tet
– Lambda Red
Eco IS150::*crtI* bgl::*cat-sacB* /pSIM5-tet
+ *crtEB* (PCR product)
Eco gsp::*crtZY* /pSIM5-tet
+ gspC::*dhfr* (PCR product)
– Plate cells Lambda Red
Eco IS150::*crtI* bgl::*cat-sacB* /pSIM5-tet + *crtEB*
Plate on sucrose + tet
Eco gsp::*crtZY* /pSIM5-tet + gspC::*dhfr*
Plate on tmp
- Restreak transductants lambda Red – leave in RT over the weekend
Eco IS150::*crtI* bgl::*cat-sacB* /pSIM5-tet + *crtEB*
Plate on sucrose + tet
Eco gsp::*crtZY* /pSIM5-tet + gspC::*dhfr*
Plate on tmp

### Week: 4/9–10/9

– Colony PCR and ran gel electrophoresis for validation of insertion. Sent for sequencing.
– Transformed BL21(DE3)\* with sequence verified CsADH2946 plasmid. Large scale expression and cell harvest.

![](https://static.igem.org/mediawiki/2017/a/a9/T--Uppsala--notebook_gel2.png)

### Lambda red integration:

– Start overnight cultures of Lambda Red transformants
Eco IS150::*crtI* bgl::*crtEB* /pSIM5-tet
Grow in LB + tet in 30 °C (1.5 ml, will also use for transduction)
Eco gsp::*crtZY* gspC::*dhfr*
Grow in LB + tmp in 37 °C (1.5 ml, will also use for P1 lysate)
– Make P1 lysate (put in fridge after lysis)
Eco gsp::*crtZY* gspC::*dhfr*
Freeze overnight cultures of lambda Red transformants
Eco IS150::*crtI* bgl::*crtEB* /pSIM5-tet
Eco gsp::*crtZY* gspC::*dhfr*

### Week: 11/9–17/9

- Purification of CsADH2946 on IMAC

### Lambda red integration:

– Screening of *crtEB* clones, run screening PCR, gel analysis

### Week: 18/9–24/9

– SDS-PAGE of CsADH2946 IMAC fractions.
– Changed buffer and concentrated pooled enzyme fractions.
– Activity measurements of CsADH2946.
– Created BioBricks of UGTCs2 by inserting it into the linearised iGEM plasmid pSB1C3-J04500 using Gibson Assembly.

### Lambda red integration:

– Screening of *crtEB* clones
Wrong band size! Redo lambda red.

### Week: 25/9–1/10

– Colony PCR of UGTCs2, gel electrophoresis and send for sequencing.
– Transformation into BL21(DE3)\* for expression
– Ordered more CaCCD2 construct from IDT.
– Sat overnight culture with UGTCs2 and performed expression testing with different concentrations of IPTG.
– Harvested cells and performed IMAC purification on ÄKTA. Ran fractions on SDS-gel. No significant bands could be seen.

![](https://static.igem.org/mediawiki/2017/6/6e/T--Uppsala--notebook_gel3.png)

### Week: 2/10–8/10

– Inserted construct of CaCCD2 into plasmid with Gibson Assembly.
– Colony PCR of CaCCD2, gel electrophoresis and send for sequencing.
– Expression testing of UGTCs2 and CaCCD2 with different concentrations of IPTG. SDS-PAGE on the crude lysates.

### Lambda red integration:

– Start cultures for Lambda Red
Eco IS150::*crtI* bgl::*cat-sacB* /pSIM5-tet
– Lambda red
Eco IS150::*crtI* bgl::*cat-sacB* /pSIM5-tet
+ *crtEB*
– Plate lambda red, incubate in RT over weekend
Eco IS150::*crtI* bgl::*cat-sacB* /pSIM5-tet
*crtEB* plate on sucrose agar (4 plates)

### Week: 9/10–15/10

– Set up pulling simulations for CsADH2946
– Combining CaCCD2 and CsADH2946 into one BioBrick using 3A assembly.
– Sent for sequencing.

### Lambda red integration:

– Restreak *crtEB* clones – There are a few colonies that look reddish
16 clones on 4 LA agar plates
– Restreak crtEB transformants – red colonies.
They grow really bad, and look quite sick.
Transduce *crtYZ* into *crtEB* strain with P1
Clone 1–1 and 2–1–1.
– Restreak 6 clones of the *crtYZ* + *crtEB* transductants on tmp agar
Start overnight cultures of restreaked *crtEB* clones in 30 °C
4 clones started
– Freeze overnight cultures of restreaked *crtEB* clones in 30 °C
4 clones frozen
Restreak crtZY transductants on tmp agar
Clone 1–1 and 2–1–1
Restreak *crtZY* transductants a second time on LB agar
Clone 1–1 and 2–1–1
– Move plates to fridge or RT
Crt strains from 30 °C

### Week: 16/10–22/10

– Prep of UGTCs2, CsADH2946 and CaCCD2 for BioBrick submission.
– Assembled J04500-CaCCD2 and J04500-CsADH2946 on pSB1K3 plasmid using 3A assembly into zeaxanthin producing *E. coli* strain.
– Transformed zeaxanthin strain with the assembled plasmid and another batch with plasmid containing only CaCCD2.
– Extracted zeaxanthin from the zeaxanthin producing strain

### Lambda red integration:

– Take photos of *crtEBIZY* strains
Start overnight cultures
*crtEBIZY* strains (LB, 30 °C)
– Freeze overnight cultures
*crtEBIZY* strains (LB, 30 °C)

### Week: 23/10–29/10

– Large scale expression of zeaxanthin strain containing CaCCD2 + CsADH2946.
– Assembled J04500-CaCCD2-J04500-CsADH2946 and J04500-UGTCs2 on pSB1A3 using 3A assembly into our zeaxanthin producing *E. coli* strain.
– TLC comparing cultures of different zeaxanthin strain transformations (Zeaxanthin strain only, zeaxanthin strain with CaCCD2, zeaxanthin strain with CaCCD2 + CsADH2946 and zeaxanthin strain with CaCCD2 + CsADH2946 + UGTCs2).

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# Team:Uppsala/Experiments

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* [Home](https://2017.igem.org/Team%3AUppsala)
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  + [Team](https://2017.igem.org/Team%3AUppsala/Team)
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* Project
  + [Description](https://2017.igem.org/Team%3AUppsala/Description)
  + [Design](https://2017.igem.org/Team%3AUppsala/Design)
  + [Crocin Pathway](https://2017.igem.org/Team%3AUppsala/CrocinPathway)
  + [Zea-Strain](https://2017.igem.org/Team%3AUppsala/Zea-Strain)
  + [Modeling](https://2017.igem.org/Team%3AUppsala/Model)
  + [Parts](https://2017.igem.org/Team%3AUppsala/Parts)
* Human Practices
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  + [Ethics-Gold HP](https://2017.igem.org/Team%3AUppsala/HP/Gold_Integrated)
  + [Engagement](https://2017.igem.org/Team%3AUppsala/Engagement)
  + [Market Analysis](https://2017.igem.org/Team%3AUppsala/Entrepreneurship)
* Results
  + [Results](https://2017.igem.org/Team%3AUppsala/Results)
  + [Demonstrate](https://2017.igem.org/Team%3AUppsala/Demonstrate)
  + [Medals](https://2017.igem.org/Team%3AUppsala/Medals)
* [Portfolio](https://2017.igem.org/Team%3AUppsala/Portfolio)
* Extended Work
  + [Chipgineering2.0](https://2017.igem.org/Team%3AUppsala/Hardware)
  + [InterLab](https://2017.igem.org/Team%3AUppsala/InterLab)
  + [Improve](https://2017.igem.org/Team%3AUppsala/Improve)
  + [Contributions](https://2017.igem.org/Team%3AUppsala/Contribution)
* [Safety](https://2017.igem.org/Team%3AUppsala/Safety)

**3A Assembly**

### 1. Digestion

A 25 µL master mix was prepared for both of the inserts. The backbone plasmid was already precut, but to cut backbone plasmids EcoRI-HF and PstI would be used (the same volume).

**Enzyme Master Mix for the first insert:**

|  |  |
| --- | --- |
| NEB Buffer 2 | 5 µL |
| EcoRI-HF | 0.5 µL |
| SpeI | 0.5 µL |
| Milliq | 19 µL |

4 µL of the master mix was then mixed with 4 µL of the enzyme.

**Enzyme Master Mix for the second insert:**

|  |  |
| --- | --- |
| NEB Buffer 2 | 5 µL |
| XBaI | 0.5 µL |
| PstI | 0.5 µL |
| Milliq | 19 µL |

4 µL of the master mix was then mixed with 4 µL of the enzyme.

Both digestion mixtures were digested at 37 °C for 30 minutes.
The mixtures were then left at 80 °C for 20 minutes to heat kill the enzymes.
These steps were performed in a PCR machine.

### 2. Ligation

In this step it is important to have equimolar amount of cut plasmid backbone and digest product. In our case the length of everything was very similar, therefore the same volumes could be used, however that can always be done. The total reaction volume is 10 µL

**Ligation Mixture:**

|  |  |
| --- | --- |
| Plasmid backbone | 2 µL |
| Enzyme insert cut at E and S | 2 µL |
| Enzyme insert cut at X and P | 2 µL |
| T4 DNA Ligase Buffer | 1 µL |
| T4 DNA Ligase | 0.5 µL |
| MilliQ | 2.5 µL |

The mixture was then ligated at 16 °C for 30 minutes.
Another heat kill around at 80 °C for 20 minutes was then performed.

For the transformation step see the [Phusion PCR, Gibson Assembly and Transformation](#phusion) protocol.

**Phusion PCR, Gibson assembly and Transformation**

### 1. Phusion PCR

Total reaction volume 50 µL
Amount of plasmid per reaction ~30 pg

**Phusion PCR reaction mixture:**

|  |  |
| --- | --- |
| Plasmid | …. µL (~30 pg) |
| 5X Phusion HF Buffer | 10 µL |
| dNTP (10 mM) | <1 µL/td> |
| Reverse Primer (10 µM) | 2.5 µL |
| Forward Primer (10 µM) | 2.5 µL |
| Phusion DNA pol | 0.5 µL |
| DMSO | 1.5 µL |
| H2O | up to 50 µL |
| Total volume | 50 µL |

**PCR reaction conditions:**

|  |  |
| --- | --- |
| 1) Initial denaturation: | 98 °C for 30 seconds |
| 2) Denaturation in following cycles: | 98 °C for 10 seconds |
| 3) Primer annealing: | Temperature gradient between 58–65 °C for 30 seconds |
| 4) Extension: | 72 °C 1.5 minutes |
| 5) Final DNA synthesis: | 72 °C for 5 minutes |

Steps 2–4: 25 cycles
Lid temperature at 105 °C

### 2. DpnI Treatment of the PCR Fragment

To the 50 µL PCR reaction mix from step 1 add:

2.4 µL “FastDigest” DpnI
6 µL 10X “FastDigest” Buffer

1) Incubate reaction mixture at 37 °C for 1 hour
2) Purify the DNA in the mixture with “PureLink Quick PCR Purification Kit” (Invitrogen). Milliq was used instead of TE buffer when eluting the DNA from the spin column in the final step of the DNA purification. The optional washing step was skipped
3) Measure the DNA concentration on Nanodrop in units of ng/µL. 2 µL was used for each measurement
4) Store the DNA at -20 °C until further use

### 3. Gibson Assembly Reaction

1. Mix PCR fragment from the plasmid and the Gibson fragment containing the insert. The total volume is 20 µL:

Mix i) the PCR fragment made from the plasmid and ii) the Gibson fragment containing the gene of interest. Total volume of the Gibson reaction mix is 20 uL :

|  |  |
| --- | --- |
| Plasmid PCR fragment | .… µL (~0.02 pmol) |
| Gibson fragment | .… µL (~0.06 pmol) |
| Gibson Assembly Master Mix (2x) | 10 µL |
| H2O | up to 20 µL |
| Total volume | 20 µL |

2. Incubate the mixture at 50 °C for 1 hour

### 4. Transformation

1. Take out competent cells (we used *Escherichia Coli* TOP10 for cloning and *E. coli* BL21 (DE3\*) for protein expression from Invitrogen) from the -80 °C and thaw them on ice from approximately 15 minutes.
2. Add 2 µL of the Gibson mixture/Ligation mixture to the competent cells or ~7.5 ng of gBlocks.
3. Incubate on ice for 30 minutes.
4. Incubate at 42 °C for 30 seconds (water bath).
5. Incubate on ice for 5 minutes.
6. Add 200 µL of LB to the mixture and incubate it at 37 °C with 200 rpm for 1 hour.
7. Plate the mixtures on chloramphenicol plates (200 µL 1:1 dilution and 200 µL 1:100 diluted with LB). Beads were used to spread the mixture on the plates (10–20 beads).
8. Incubate plates overnight at 37 °C.

### Electroporation

1. Grow overnight culture in 5 mL LB media.
2. Wash with 1 mL 10 % glycerol 5 times in cold room.
3. Resuspend in 250 µL 10% glycerol. This is enough for 10 electroporation transformations.
4. Put 1 µL plasmid (~10 ng/µL) in 25 µL cells. Transfer to a cold electroporation cuvette.
5. Electroporate with a 1.8 kV pulse. Immediately add 300 µL SOC media. Incubate in 37°C with 180 rpm shaking for 1h. Plate on appropriate antibiotic agar plate.

**Colony PCR and Gel Electrophoresis**

### 1. Colony PCR

Total reaction volume 20 µL.

**DreamTaq PCR Reaction Mixture:**

|  |  |
| --- | --- |
| 10X Green DreamTaq Buffer | 2 µL |
| dNTP Mix, 10 mM | 0.4 µL |
| Forward Primer | 0.1–1.0 µM (~0.5 µL) |
| Reverse Primer | 0.1–1.0 µM (~0.5 µL) |
| Template DNA | 10 pg – 1 µg |
| DreamTaq DNA Polymerase | 0.1 µL |
| Milliq | up to 20 µL |

A master mix containing everything put the DNA polymerase was made. Enzyme was then added to each PCR tube.

**PCR reaction conditions:**

|  |  |
| --- | --- |
| 1) Initial denaturation: | 95 °C for 3 minutes |
| 2) Denaturation in following cycles : | 95 °C for 30 seconds |
| 3) Primer annealing: | 56 °C for 40 seconds |
| 4) Extension: | 72 °C 1 minute |
| 5) Final DNA synthesis: | 72 °C for 5 minutes |

Steps 2–4: 32 cycles
Lid temperature at 105 °C

### 2. Gel electrophoresis

**Preparation of agarose for gel electrophoresis:**

1. Mix 0.5 % TEB with agarose so that the amount of agarose is 0.8 %
2. Microwave the mixture until it starts boiling. DO NOT PUT THE LID ON!
3. Store at 65 °C until further use

**Preparation of gels**

1. Take 50 mL of agarose per gel into a separate bottle
2. Add 5 µL of SyberSafe per gel into the agarose
3. Pour the agarose into the cassette, put the comb in and let it polymerize for ~30 minutes
4. Put the gel into the electrode chamber and fill it up with 0.5 % TEB buffer
5. Load 12 µL of PCR product into the wells and add 5 µL of 1 kb Generuler
6. Run the gel at 100 V for 30–60 minutes

**Expression, IMAC Purification, SDS-PAGE analysis & Activity Measurements**

### Expression

1. Transform plasmid in BL21 (DE3\*) *E. coli* cells to use for expression.
2. Inoculate three colonies from transformation plate into 50 mL LB with 30 µg/ml chloramphenicol. Make a negative control without any colony and grow overnight.
3.Take 20 mL of culture in 2 L LB with 30 µg/mL chloramphenicol to make expression cultures, divided into two 1 L E-flasks. Grow at 37 °C 110 rpm for about 3 hours until 0.6 < OD600 < 0.8. Take out negative control sample that is not induced. Add 0.25–1 mM IPTG to induce protein expression and grow at 37 °C 110 RPM for about 4–6 hours.
4. Divide culture into 4 tubes and centrifuge culture at 5000 rpm for 30 min. Discard supernatant and resuspend each pellet in about 20 mL LB. Pour over to tw0 50 mL Falcon tubes and centrifuge 5 min at 7000 rpm. Discard the supernatant. Flash freeze pellet in liquid nitrogen and store at -80 °C.

### IMAC Purification

**Buffer A, 1 L**
The buffer was made with MilliQ H2O. NaOH was added until pH 7.4 and sterile filtered on 0.2 µm filter.

| Buffer component | Concentration |
| --- | --- |
| NaH2PO4/NA2HPO4 | 20 mM |
| NaCL | 500 mM |
| Imidazole | 20 mM |
| beta-ME (added on day of chrom.) | 5 mM |
| Protease inhibitor pill EDTA-free(added on day of chrom.) | 30% of pill |

**Buffer B, 0.5 L**
The buffer was made with MilliQ H2O. Phosphoric acid was added until pH 7.4 and sterile filtered on 0.2 µm filter.

| Buffer component | Concentration |
| --- | --- |
| NaH2PO4/NA2HPO4 | 20 mM |
| NaCl | 500 mM |
| Imidazole | 500 mM |
| beta-ME (added on day of chrom.) | 5 mM |
| Protease inhibitor pill (added on day of chrom.) | 30% of pill |

(Day of purification)
1. Thaw the cells (2 pellets) and add 10 mL buffer A to each pellet. Add 20% of EDTA-free protease inhibitor pill to each pellet. Add 500 µL lysozyme (10 mg/ml) and 2 µL DNAse I (238 units/µL). Put on stirring at 4 °C for 40 min.
2. Use a cell disruptor to lyse the cells and pellet for 1 hour at 3800 rfc and run the supernatant through a 0.2 µm vacuum filter.
3. Add the supernatant to Ni2+-agarose column on an ÄKTA protein purification system. 3 mL fractions were collected.
4. Wash the injection tubes by running buffer A for some minutes. Wash column with buffer B and equilibrate with buffer A. Wash with 50–100 mL buffer A until A280 hit baseline. Start eluting gradient to 100 % buffer B in 60 min. Collect 3 mL fractions.
5. **SDS-PAGE analysis**
a. Run the fractions containing protein on SDS-PAGE to identify fractions holding the desired protein.
b. Load premade gels (4–20 % SDS) with 20 µL sample (10 µL protein sample and 10µL of Laemmli buffer) as well as 6 µL PageRuler-prestained ladder 10-180 kDa (ThermoFisher) and run the gel in running buffer at 200 V for about 30 min.
c. Stain gels in Coomassie blue, destain and document the result.
6. Pool fractions containing purified protein and perform buffer exchange to PBS+500 mM NaCl using Amicon Ultra Centrifugal filters 10 kDa. This should be done before performing activity measurements.

### Activity measurements

1. Before activity measurements of the purified enzyme buffer exchange to PBS+500 mM NaCl should be performed using Amicon Ultra Centrifugal filters 10 kDa.
2. The buffers should be freshly prepared.
3. Use plate reader to performed the measurements.
4. The final volume in each well was 75 µL. 65 µL + 10 µL of protein (enzyme).
5. Substrates were dissolved in DMSO stock solution.
6. Negative control (substrate without protein) and positive control (product without protein) were used.
7. Buffer without protein, substrate or product was used as a blank.
8. Absorbance spectra was in the 200–700 nm range and measurements were taken at 1–15 min intervals until activity had been observed.

**CsADH2946**
Protein concentration was varied in each well, see specifics for the experiment [here](https://2017.igem.org/Team%3AUppsala/CrocinPathway#step2)

| Buffer component | Concentration |
| --- | --- |
| KCl | 100 mM |
| NAD+ | 0.67 mM |
| 2-Mercaptoethanol | 10 mM |
| Crocetin dialdehyde | 150 µM |
| Crocetin (positive control only) | 150 µM |
| Tris-HCl, pH 8.0 | 100 mM |

**λ red recombination**

**P1 LB medium**

Standard LB + 10-25 mM MgCl2, 5 mM CaCl2, 0.1-0.2% glucose

**Na-citrate LB medium”**

Standard LB + 100 mM Na-Citrate (pH 5.5)

**Lysate preparation**

1. Grow an overnight culture of the donor strain (with selection if needed).

2. Inoculate 2 x 4 ml P1 LB medium in 50 ml tubes with 50µl culture per tube.
DO NOT ADD ANTIBIOTICS!
Grow shaking with aeration at 37 ˚C for 1-1.3 h. When the cells are in early log phase (slightly turbid, but noticeable growth), add 100 µL of P1 phage lysate to one of the cultures, continue growing at 37 ˚C. Monitor for 1–4 h, and when the culture has lysed, transfer the cells in the other tube to 2 ml tubes, spin down the cells and resuspend them in 200 µl P1 LB. Add the resuspended cells to the lysed tube and continue growing at 37 ˚C. Monitor for 1–4 h until the culture has lysed.

3. Transfer lysate to 2 ml tubes, and add 100 µl of chloroform to the lysate and vortex 10 sec. Centrifuge away the debris (13000 rpm, 2 min) and transfer the supernatant to a fresh tube. Add a few drops of chloroform and store at 4 ˚C.

**P1 transduction**

1. Grow recipient overnight in P1 LB (1 ml culture per transduction).

2. Add 75 μl donor P1 lysate to 400 μl culture of the recipient strain, mix, and incubate the tubes in a 37 ˚C water bath for 30 min.

6. Spin cells at 6000 rpm for 5 min, remove supernatant carefully and resuspend the pellet in 100 µL Na-citrate LB, vortex well to disperse cells, and plate all of it on an appropriate antibiotic-containing plate.

8. You should get anywhere from ~ 10 to 2000 colonies. These colonies are growing on a plate that is covered with P1 phage, so use a toothpick to touch the top of a few colonies and re-streak on new selective plates for isolated colonies.

9. Test a colony from each re-streak for the presence of the mutant gene you intended to transduce using PCR.

**P1 transduction**

*E. coli*:
DA24100 Escherichia coli K12 MG1655 /pSIM5-Tet Lambda Red system TetR
This strain carries the pSIM5-tet plasmid, see above. Grow at 30°C, 12.5 μg/ml tetracycline.

1. Grow strain O/N at 30°C in Salt Free LB (with appropriate antibiotic).
2. Dilute 1:100 in 30°C pre-warmed Salt Free LB (with appropriate antibiotic for pSIM5/pSIM5-Tet/pSIM6/pSIM-ctx) in a 500ml E-flask. 50 ml is enough for 4-8 electroporations.
3. Grow at 30°C shaking until OD600 ≈ 0.3-0.4 (1.5~2 hrs).
4. To induce red expression, move the flask to a 42°C shaking waterbath, incubate with slow shaking (110 rpm) for 15 minutes.
5. Cool cells on ice for at least 10 minutes. Swirl carefully to cool cells.
6. Transfer cells to cold 50 ml tubes and spin down the cells (4500 rpm, 8 min., 4°C).
7. Remove as much as possible of the supernatant and resuspend the cells in 100 µl
ice cold sterile water using a 10 µl loop. Add 12 ml ice cold water and pipette up and down a few times (using the pipette-boy). Do not vortex! Pellet the cells at 4500 rpm, 6-10 min, 4°C and repeat the wash one more time.
8. Resuspend the cells in ice-cold 10% glycerol (as small volume as you need, ~300 µl).
9. Mix 40 μl cells and DNA in an electroporation cuvette on ice (2-8 µl de-salted PCR product or up to half of what you get from a normal 20 μl PCR reaction, 100-500 ng.
10. Electroporate (2.5 kV 200 Ω, 25 µF). Immediately resuspend the cells in 1 ml 42°C SOC or SOB and transfer to 10 ml plastic tubes.
11. Optional: Incubate the cells in a 42°C water bath for 10 minutes.
12. Let the cells recover for at least 3 hrs or overnight in a 30°C shaking incubator.
13. Plate on appropriate selective medium, incubate the plates at 30°C O/N.
14. Restreak colonies on selective medium at 42°C. Test for loss of the plasmid. (Consider freezing a clone that still contains the pSIM-plasmid.)

**Extracting zeaxanthin from E. coli strain and TLC**

1. Pick a colony and grow in LB overnight in 30°C in shaking incubator.
2. For an analytical extraction, spin down 10 mL cells and lyse with 1 mL acetic acid.
3. Pour 1 mL toluen and 1 mL H2O into the tube to create a two-phase separation. Spin down. Take the upper organic phase into a new tube and evaporate under vacuum until a yellow pigment powder is visible. Resolve in 10 uL toluen and measure the absorbance spectra on nanodrop.
4. Run thin layer chromatography (TLC) on a silica coated aluminum plate with 90% chloroform and 10% ethanol as mobile phase.

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# Team:Uppsala/Results

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* [Home](https://2017.igem.org/Team%3AUppsala)
* Team
  + [Team](https://2017.igem.org/Team%3AUppsala/Team)
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* Project
  + [Description](https://2017.igem.org/Team%3AUppsala/Description)
  + [Design](https://2017.igem.org/Team%3AUppsala/Design)
  + [Crocin Pathway](https://2017.igem.org/Team%3AUppsala/CrocinPathway)
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  + [Results](https://2017.igem.org/Team%3AUppsala/Results)
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* [Safety](https://2017.igem.org/Team%3AUppsala/Safety)

<!DOCTYPE html>
Results

![](https://static.igem.org/mediawiki/2017/d/d8/Uppsala-Results_Final_Header.svg)

Summary

![](https://static.igem.org/mediawiki/2017/3/30/Results_line_under_title.svg)

We successfully integrated the first five steps of the crocin pathway from FPP to zeaxanthin into the *E. coli* chromosome. The result is a *E. coli* strain expressing zeaxanthin. We have created sequence verified BioBricks of our enzymes in the extended crocin pathway: CaCCD2 ([BBa\_K2423005](https://2017.igem.org/Team%3AUppsala/Parts)), CsADH2946 ([BBa\_K2423007](https://2017.igem.org/Team%3AUppsala/Parts)) and UGTCs2 ([BBa\_K2423008](https://2017.igem.org/Team%3AUppsala/Parts)). We have also characterized these enzymes with experiments and simulations.  to purify and confirm activity of CsADH2946 as well as measuring the kinetic parameters of the enzyme.

![](https://static.igem.org/mediawiki/2017/1/18/Uppsala-ZeaPlate.png)

Figure 1. Top: Wild-type *E. coli*. Bottom: Zeaxanthin producing *E. coli* strain with 5 genes inserted into the chromosome.

We created and combined the zeaxanthin producing strain with a plasmid containing the extended crocin pathway which gave us an *E. coli* strain including the entire production pathway from FPP to crocin. **In the end, we were able to identify, create and extensively characterize the pathway for crafting crocin**.

![](https://static.igem.org/mediawiki/2017/0/07/Results_title_footer.svg)

Chromosomal integration:

Farnesyl Pyrophosphate (FPP) → Zeaxanthin

![](https://static.igem.org/mediawiki/2017/3/30/Results_line_under_title.svg)

* Successfully integrated five genes from FPP to zeaxanthin into the [chromosome](https://2017.igem.org/Team%3AUppsala/Zea-Strain) of *E. coli*.
* Successfully transformed the crocin pathway into the [zeaxanthin producing strain](https://2017.igem.org/Team%3AUppsala/Zea-Strain)
* Extracted zeaxanthin from the [zeaxanthin producing strain](https://2017.igem.org/Team%3AUppsala/Zea-Strain)

![](https://static.igem.org/mediawiki/2017/0/07/Results_title_footer.svg)

Step 1: [CaCCD2](https://2017.igem.org/Team%3AUppsala/CrocinPathway)

Zeaxanthin → Crocetin dialdehyde

![](https://static.igem.org/mediawiki/2017/3/30/Results_line_under_title.svg)

* [Biobrick](https://2017.igem.org/Team%3AUppsala/Parts) – Coding for the enzyme CaCCD2 with his-tag and lac-inducible promoter, characterised with correct sequencing!
* Homology [model](https://2017.igem.org/Team%3AUppsala/Model)
* [Molecular dynamics](https://2017.igem.org/Team%3AUppsala/Model) – the model was stable!
* Successfully combined with pathway and transformed into [zeaxanthin strain](https://2017.igem.org/Team%3AUppsala/Zea-Strain)!

![](https://static.igem.org/mediawiki/2017/0/07/Results_title_footer.svg)

Step 2: [CsADH2946](https://2017.igem.org/Team%3AUppsala/CrocinPathway)

Crocetin dialdehyde → Crocetin

![](https://static.igem.org/mediawiki/2017/3/30/Results_line_under_title.svg)

* [Biobrick](https://2017.igem.org/Team%3AUppsala/Parts) – Coding for the enzyme CsADH2946 with his-tag and lac-inducible promoter, characterised with correct sequencing and activity!
* Homology [model](https://2017.igem.org/Team%3AUppsala/Model)
* [Molecular dynamics](https://2017.igem.org/Team%3AUppsala/Model) – the model was stable!
* [Steered Molecular Dynamics (pulling)](https://2017.igem.org/Team%3AUppsala/Model) – estimated Kd(=4.9321 µM)
* [Measurement of Km](https://2017.igem.org/Team%3AUppsala/Model#KM) (=20.7842 µM)
* [Chromatogram and SDS-PAGE gel](https://2017.igem.org/Team%3AUppsala/CrocinPathway) showing our protein successfully expressed and purified
* [Activity](https://2017.igem.org/Team%3AUppsala/CrocinPathway) against the conversion of crocetin dialdehyde to crocetin
* Successfully combined with pathway and transformed into [zeaxanthin strain](https://2017.igem.org/Team%3AUppsala/Zea-Strain)!

![](https://static.igem.org/mediawiki/2017/0/07/Results_title_footer.svg)

Step 3: [UGTCs2](https://2017.igem.org/Team%3AUppsala/CrocinPathway)

Crocetin → Crocin

![](https://static.igem.org/mediawiki/2017/3/30/Results_line_under_title.svg)

* [Biobrick](https://2017.igem.org/Team%3AUppsala/Parts) – Coding for the enzyme [UGTCs2](https://2017.igem.org/Team%3AUppsala/Improve) with his-tag and lac-inducible promoter, characterised with correct sequencing!
* Homology [model](https://2017.igem.org/Team%3AUppsala/Model)
* [Molecular dynamics](https://2017.igem.org/Team%3AUppsala/Model) – the model was stable!
* Successfully combined with pathway and transformed into [zeaxanthin strain](https://2017.igem.org/Team%3AUppsala/Zea-Strain)!

![](https://static.igem.org/mediawiki/2017/0/07/Results_title_footer.svg)

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