iGEM 2019 — Team UiOslo_Norway

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# Team:UiOslo Norway/Notebook

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☰

UiOslo

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| 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| 28 | 29 | 30 | 31 |  |  |  |

# Notebook

Welcome to our notebook!
Click on the calendar if you are curious to see what we did each week.

# Week 21

Who worked: Simen, Linnea

### Transformation

- E. coli (DH5a) transformed with pBAD

- Used LB media with ampicillin

### Miniprep

- Miniprep of transformed cultures

- RESULT: concentrations of pBAD --> Sample 1: 52ng/ul, Sample 2: 54ng/ul

### PCR

- PCR of pBAD, Sample 2 (from miniprep)

- Annealing temperatur: 64°C

- RESULT: No product on gel.Thinking of doing Gradient PCR to find right annealing temperature

# Week 22

Who worked: Simen, Linnea

### Gradient PCR

- Trying to find optimal PCR temperatures between 59°C - 69°C

- 8 samples made with different annealing temperatures from Sample 2, pBAD (made week 21)

- RESULT: Samples : 7 (68.3°C) and 8 (69°C) looked good on the gel.

# Week 23

Restricted lab access

# Week 24

Restricted lab access

# Week 25

Who worked: Simen, Alec, Prabin

### DpnI digestion

- Digestion of all gradient PCR products 1-8 (made Week 22)

- DNA was run on a gel after digestion with DpnI

- RESULT: concentration sample 7 (36.5ng/ul)

### Gel Purification

- Purification of gel

### Gibson assembly

- 3 gibson assemblies, putting pBAD together with each of our three genes: crtI, crtE and crtB

- Used the pBAD backbone from sample 7

- Had two controls, one with our pBAD vector from Sample 7, together with Gibson mix and the other one with only pBAD and water

|  |  |  |
| --- | --- | --- |
| Rxn 1 | Concentration | Amount Needed |
| pBAD | 36.5ng/ul | 3ul |
| CrtI | 28ng/ul | 3ul |
| 2xGibson mix | -------- | 6ul |

|  |  |  |
| --- | --- | --- |
| Rxn 2 | Concentration | Amount Needed |
| pBAD | 36.5ng/ul | 3ul |
| CrtE | 17ng/ul | 2ul |
| 2xGibson mix | -------- | 5ul |

|  |  |  |
| --- | --- | --- |
| Rxn 3 | Concentration | Amount Needed |
| pBAD | 36.5ng/ul | 3ul |
| CrtB | 47ng/ul | 1ul |
| 2xGibson mix | -------- | 4ul |

- RESULTS:

### Making competent cells

RESULT: succesful

### Transformation

- Transformation of crtB, crtE and crtI into DH5a

- RESULT: number of colonies --> crtB (14 colonies), crtE (5 colonies) and crtI (2 colonies)

### Colony PCR

- Only sample 7 and 8 were done gel extractions on

### Colony PCR

- Colony PCR with colonies from gibson assembly

- Petri dish with numbered grids was added colonies from the Gibson reaction

- Colonies with pBAD-crtB, pBAD-crtE and pBAD-crtI were added to plate in grid 1-14, 15-19 and 20-21, respectively

- Remaining cells, after colonies were added to grid, were done colony PCR on

### Liquid culture

- Made liquid cultures of colonies: 7 and 14 (crtB), 15 and 19 (crtE) and 20 (crtI) for the next day

- Gave the samples new names. 7= crtB1, 14=crtB2, 15=crtE1, 19=crtE2, 20=crtI

### Miniprep

- Miniprep preformed on inoculated samples from day before.

- RESULT: concentrations -- > crtB1 (121.2ng/ul), crtB2(118.9ng/ul), crtE1(144.5ng/ul), crtE2(125.4ng/ul), crtI(133.2ng/ul)

### Sequencing

- crtB1, crtB2, crtI, crtE1 and crtE2 sent for sequencing

- RESULT: crtB1 and crtI had the correct sequence and were kept for SDS-PAGE (week 26), CrtE1 and E2 had faults in the sequence and were thrown away

### Expression Test

- Made expression cultures of colonies crtE1 and 2, crtB1 and 2, and crtI

- Induction with 0.1% final concentration of Arabinose

- RESULT: the expression seemed to have been a failure, with no difference between the induced and the non-induced cultures. The expression test needs to be repeated. Next time the cultures should contain glucose? Also, does DH5a consume arabinose so that the expression won’t work unless we use another strain?

# Week 26

Who worked: Simen, Alec, Marthe

### Miniprep

- On colonies 16-18 (crtE)

### Sequencing

- Miniprepped DNA from colonies 16-18 were send for sequencing

# Week 27

Who worked: Bianca, Simen, Alec

### Transformation

- Transformation of pBAD-crtI, pBAD-crtE and pBAD-crtB into DH5a cells

- RESULT: several colonies on plate

### Preculture

- Made pre-cultures on crtI, crtE and crtB

# Week 28

Who worked: Alec, Bianca

### Cell Lysis

- Lysis of cells by sonication

### SDS-PAGE

- Run proteins on SDS gel from crtI, crtE and crtB from previous week

# Week 29

Who worked: Prabin, Linnea, Alec, Simen

### Preculture

- Setting up new precultures to test crtE and crtI

### Expression culture

- Making expression culture from preculture of crtE and crtI

- 0.2% Glucose, 100ug/ml ampicillin

- Two different concentrations of arabinose: 0.2% and 0,02%

- RESULT: crtE (OD600 = 0.7968), crtI (OD600 = 0.847)

### Cell lysis

- Cell lysis of cells in expression culture by Sonication

### SDS-PAGE

- SDS-PAGE of lysed cells after expression cultures

### Expression test

- Expression test on crtI

Done glucose free, with 0.1ng/ml ampicillin

- Two different concentrations of arabinose: 0.2% and 0,02%

- Checked OD600 after 3 and 3.5hrs.

- RESULT: 3hrs (OD600 = 0.3359), 3.5hrs (OD600 = 0.5038)

### Cell lysis

- Cell lysis of cells in expression culture by Sonication

### SDS-PAGE

- The lysed cells were run on an SDS-gel

- The samples from the last expression test, were made dilutions on and run on a SDS-gel

- The dilutions were: 1x, 2x, 3x, 5x, 10x, 20x, 50x, 100x

# Week 30

Who worked: Alec, Prabin

### Preculture

- Precultures of crtE, crtI and crtB.

- Used low Glucose LB media

### Expression test

- Made expression culture from preculture

Done with 0.1ng/ml ampicillin

- Five different concentrations of arabinose, 0.05%, 0.1%, 0.2%, 0.5% and 1%

- DEVIATION: Shaker stopped overnight

- RESULT: after 3.5hrs, 2% arabinose -- > crtB (OD600 = 0.872), crtE (OD600 = 0.913), crtI (OD600 = 0.867)

### SDS-PAGE

- Making test gel for testing all samples from expression test

- RESULT: Something went wrong and the bands were barely visible on the gel. Will run SDS again without dilutions

### SDS-PAGE

- SDS-gel with samples from expression test run without dilutions, only 2% arabinose samples.

# Week 31

Lab closed due to building maintenance

# Week 32

Who worked: Alec, Simen, Prabin

#### Preculture

- Preculture of crtE, crtB and crtI, with ampicillin

#### Expression Test

- Made expression culture from preculture

- Three different concentrations of arabinose, 0.5%, 1%, 2%

- RESULT: after 3hrs -- > crtB (OD600 = 0.5406), crtE (OD600 = 0.6145), crtI (OD600 = 0.6248).

after 24hrs -- > (see table for OD600)

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| % Arabinose | 0% | 0.5% | 1% | 2% |
| crtE | 1.1 | 2.1 | 2.0 | 1.8 |
| crtB | 2.6 | 2.1 | 2.2 | 1.9 |
| crtI | 2.4 | 2.3 | 2.1 | 2.0 |

# Week 33

Who worked: Marthe, Simen, Alec, Prabin

### Colony PCR

- Colony PCR of colony 17 and 18 crtE and colony 1 and 2 crtB

- RESULT: Amplification was successful. crtE also had significant amplification of unspecific product

### DpnI Digestion

- DpnI Digestion on DNA product from colony PCR

### DNA Purification

- Purification of DNA product from DpnI digestion.

- Purification of product 2 (crtE) using the Monarch DNA Purification Protocol

- Purification of product 4 (crtB) using Mini Elute PCR Purification Kit

- RESULT: concentrations -- > crtE (3.3ng/ul), crtB (141.8ng/ul)

### Cell Lysis

- Cell Lysis by sonication

### Gradient PCR

- Setting up gradient PCR to improve yield of crtE

- Annealing temperatures from 60 - 70 °C

- RESULT: High temperatures had less unspecific binding. Next time, we will use 70°C annealing temperature for crtE

### DpnI digestion

- DpnI digestion of crtE

### DNA Purification

- DNA purification of crtE on gel with monarch kit

- RESULT: concentration -- > crtE (33ng/ul)

### Gibson Assembly

- Gibson assembly of crtB into pBAD-crtE

|  |  |  |
| --- | --- | --- |
|  | Concentration | Amount Needed |
| pBAD-crtE | 33 ng/ul | 3 ul |
| CrtB | 141 ng/ul | 1 ul |
| 2xGibson mix | -------- | 4 ul |

- RESULT: Multiple colonies. Will check them with colony PCR.

# Week 34

Who worked: Marthe, Simen, Alec, Prabin

### Colony PCR

- Colony PCR of 9 crtEB colonies

- RESULT: colony 6 looked good

### Liquid culture

- Inoculation of colony 6, crtEB from grid plate after colony PCR

### Gradient PCR

- Gradient PCR of crtEB and crtE

### Mini-Prep

- Miniprep of inoculated culture from crtEB, colony 6

- RESULT: concentration -- > crtEB (98.5ng/ul)

### NdeI Digestion

- NdeI digestion of crtEB and crtE after gradient PCR

### PCR

- PCR of NdeI digested crtEB and crtE, as well as colonies 1-7 and 16-18 of crtEB from Gibson assembly

### PCR

- Repeat last PCR

### Transformation

- Transformation crtEB into DH5a

- RESULT: 24 colonies

### Colony PCR

- Colony PCR of colonies from Transformation

- RESULT: none of the colonies had the insert

# Week 35

Who worked: Alec, Simen , Kristin, Linnea

### PCR

- Amplification of crtB and crtE

### DpnI digestion

- DpnI digestion of PCR product

### Gel purification

- Gel purification on PCR product

Purification of crtE (Gene JETgel extraction kit) and crtB (PCR purification Kit)

-RESULT: concentrations -- > crtE vector yield (15.9ng/ul), crtB (195ng/ul)

### Gibson assembly

- Gibson assembly of crtB into the pBAD-crtE vector

|  |  |  |
| --- | --- | --- |
|  | Concentration | Amount Needed |
| pBAD-crtE | 15 ng/ul | 7.0 ul |
| CrtB | 195ng/ul | 0.6 ul |
| 2xGibson mix | -------- | 7.6 ul |

- RESULT: The Gibson assembly was successful!

### Gel purification

- Gel purification with monarch protocol

-RESULT: crtB concentration 65.0ng/ul

### PCR purification

- Purification of sample 5, crtB from PCR (from week 33)

- RESULT: concentration 195ng/ul of crtB

### Transformation

- Transformation of the crtEB , from the Gibson assembly, into DH5a cells

### Colony PCR

- Colony PCR of colonies from transformation

- Remark: 0.5ul was used instead of 0.25ul.

- Leftovers from recovery was plated on Amp-LB plate.

### Mini prep

- Miniprep of pBAD-crtEB product, colony 1, from colony PCR.

### Sequencing

- Miniprep product from colony 1 (pBAD-crtEB) sent for sequencing.

### Colony PCR

- Checking 8 colonies from the transformation.

## -------------------------HQ-part-------------------------

### Dissolve DNA pellet in water

- Dissolve pellet, from the ordered pBAD-crtEBI construct, in water

### Transformation

- Transformation of crtEBI-HQ into DH5a cells

- REMARK: HEatshock was too long

- RESULT: successful transformation

### Pre-culture

- Pre-culture of colonies from the transformation

### Mini-Prep

- Mini-prep of DNA from pre-culture

- RESULT: pink/red pellet. Concentration 87.4ng/ul

# Week 36

Who worked: Kristin, Marthe, Simen, Linnea, Alec

### PCR

- 3x different PCR reaction

- Two samples per reaction, 6 samples in total.

Rxn 1: crtEB, Rxn 2: crtI insert, Rxn 3: pBAD vector.

### Gel purification

- The products from the PCR are all run on a gel and purified.

RESULT: crtEB vector (7.7ng/ul), crtI insert (46.2ng/ul), pBAD vector (21.0 ng/ul)

### Gradient PCR

- Gradient PCR done on pBAD-crtEB vector

- Annealing temperature set from 59°C – 69°C for samples.

### DpnI Digestion

- DpnI digestion of gel purified product.

- Done on 37°C for 1h

### Gibson Assembly

- Gibson assembly of crtI insert into vector with crtEB

|  |  |  |
| --- | --- | --- |
| 1st TRY | Concentration | Amount Needed |
| CrtEB | 7.7 ng/ul | 14 ul |
| CrtI | 46.2 ng/ul | 2 ul |
| 2xGibson mix | -------- | 16 ul |

- RESULT: Assembly not successful

### Transformation

- Transformation of product from Gibson assembly, pBAD-crtEBI, into DH5a

- RESULT: Colonies on plate

### Colony PCR

- Colony PCR of 32 colonies from Transformation

Grid plate made for all colonies in the PCR reaction

### Inoculation Of Colonies

- Inoculation of colonies; 2, 6 and 10 from grid plate made with colony PCR

### Miniprep

- Miniprep of inoculated colonies

### NdeI Digestion

- NdeI digestion of miniprepped colonies: 2, 6 and 10.

- Digest run on gel

### Gradient PCR

- Gradient PCR of crtEB

- Annealing temperature set from 59°C – 69°C for samples.

## ------------------------------HQ-Part------------------------------

### PCR

- PCR of the mini prepped product (week 35)

### Gel purification

- Run the PCR product on a gel and do gel purification

- RESULT: concentration -- > crtEBI-HQ (30.5ng/ul)

### Gibson Assembly

- Cloning crtEBI into our own pBAD backbone, trying to fix leaky expression.

|  |  |  |
| --- | --- | --- |
|  | Concentration | Amount Needed |
| CrtEBI-HQ | 30.5 ng/ul | 9ul |
| pBAD | 27.0 ng/ul | 5 ul |
| 2xGibson mix | -------- | 14 ul |

### Transformation

- Transform product from Gibson assembly into DH5a cells

- DEVIATION: used 15ul Gibson solution, instead of 14 (supposed to have 1:1 relation)

- RESULT: no colonies

# Week 37

Who worked: Marthe, Simen, Kristin, Linnea, Alec

### DpnI Digestion

- DpnI digestion of amplified crtEB. (From fist PCR, preformed week 36)

### PCR Purification

- PCR purification of pBAD and crtI ()

### Gibson Assembly

- Gibson assembly of crtI and crtEB into pBAD

|  |  |  |
| --- | --- | --- |
| 2nd TRY | Concentration | Amount Needed |
| CrtEB | 38.6 ng/ul | 2.8 ul |
| CrtI | 144.2 ng/ul | 0.64 ul |
| 2xGibson mix | -------- | 3.4 ul |

- RESULT: No colonies on plate

### Gibson Assembly

- Assembly of crtEB and crtI into pBAD vector

- Making reaction with 1:3 ratio between number of inserts and backbones

|  |  |  |
| --- | --- | --- |
| 3rd TRY | Concentration | Amount Needed |
| CrtEB | 38.6 ng/ul | 2.6 ul |
| CrtI | 144.2 ng/ul | 7.2 ul |
| 2xGibson mix | -------- | 9.8 ul |

- RESULT: No colonies on plate

### PCR

- PCR of crtEB and crtI

- The annealing temperatures were: 69°C for crtEB and 63°C for crtI

- The buffers were : GC for crtEB and HF for crtI

### DpnI Digestion

- Digestion of all PCR products 1-8

### Gel Purification

- Purify products from the DpnI digestion

### Gibson Assembly

- Assembly og crtEB together with crtI pBAD

- Making reactions with a 1:3 ratio between number of inserts and backbones

|  |  |  |
| --- | --- | --- |
| 4th TRY | Concentration | Amount Needed |
| CrtEB | 17 ng/ul | 5.9 ul |
| CrtI | 19 ng/ul | 4.6 ul |
| 2xGibson mix | -------- | 10.5 ul |

- RESULT: many colonies

### Colony PCR

- PCR of 16 colonies from last gibson assembly

- Used crtEB vector as control

- Colonies boiled for 10min, 98°C

### Colony PCR

- Re-run of PCR of all colonies except colony 15

- 2min elongation time, on all colonies except nr. 15

- RESULT: None of the grid plate colonies grew up

## ------------------------------HQ-Part------------------------------

### PCR Purification

- Purification of PCR product form first PCR, week 36

### Gibson Assembly

- Cloning crtEBI into our own pBAD backbone, trying to fix leaky expression

|  |  |  |
| --- | --- | --- |
| 1st TRY | Concentration | Amount Needed |
| CrtEBI-HQ | 49.3 ng/ul | 5.6 ul |
| pBAD | 41.9 ng/ul | 3.2 ul |
| 2xGibson mix | -------- | 8.8 ul |

- RESULTAT: No colonies on plate

### Gibson Assembly

- Cloning crtEBI into our own pBAD backbone, try nr. 2

- Making reaction with 1:3 ratio between number of inserts and backbone

|  |  |  |
| --- | --- | --- |
| 2nd TRY | Concentration | Amount Needed |
| CrtEBI-HQ | 49.3 ng/ul | 6.8 ul |
| pBAD | 41.9 ng/ul | 2.4 ul |
| 2xGibson mix | -------- | 9.2 ul |

DEVIATION:

- RESULTAT: No colonies on plate

### PCR

- PCR of crtEBI-HQ and pBAD

- Annealing temperature was set to 63°C

### DpnI Digestion

- DpnI digestion of all PCR products 1-8, from last PCR

### Gel Purification

- Purification of products from DpnI digestion

### Gibson Assembly

- Cloning crtEBI into our own pBAD backbone, try nr. 2

- Making reaction with 1:3 ratio between number of inserts and backbone

|  |  |  |
| --- | --- | --- |
| 3rd try | Concentration | Amount Needed |
| CrtEBI-HQ | 47.2 ng/ul | 5.6 ul |
| pBAD | 19.2 ng/ul | 5.2 ul |
| 2xGibson mix | -------- | 10.8 ul |

- RESULT: Many colonies on plate

### Colony PCR

- PCR of 16 colonies from Gibson cloning, try nr. 3

- Used pBAD vector as control

- Colonies boiled for 10 min, 98°C

###

-

# Week 38

Who worked: Simen

### Gibson Assembly

- Gibson assembly repeated again (from week 37), 5th try.

- Use plates with arabinose

|  |  |  |
| --- | --- | --- |
| 5th TRY | Concentration | Amount Needed |
| CrtEB | 17 ng/ul | 5.9 ul |
| CrtI | 19 ng/ul | 4.6 ul |
| 2xGibson mix | -------- | 10.5 ul |

- RESULT: No colonies. We think the problem has to do with the DNA concentration. The DNA concentration has to be improved.

### Colony PCR

- PCR of colonies from 4th try of gibson assembly

### Gel Purification

-RESULT: crtEB (16.4 ng/ul), crtI (40.4 ng/ul)

### PCR

-Re-do of PCR

- PCR of crtI and crtEB

- GC buffer on crtEB and HF buffer on crtI

### PCR Purification

- Purification of PCR products

- RESULTS: crtEB (108.4 ng/ul), crtI (206.55 ng/ul)

### Gibson Assembly

- Making reaction with a 1:3 ratio between number of inserts and backbones

|  |  |  |
| --- | --- | --- |
|  | Concentration | Amount Needed |
| CrtEB | ng/ul | ul |
| CrtI | ng/ul | ul |
| 2xGibson mix | -------- | ul |

- RESULTS: colonies on gel!

## ------------------------------HQ-Part------------------------------

### PCR

- Re-do of first PCR, from week 37

- PCR of crtEBI-HQ and pBAD

### PCR Purification

- Purification of PCR products

- RESULT: crtEBI-HQ (108.4 ng/ul), pBAD (206.6ng/ul)

### Gibson Assembly

- Insetion of crtEBI into pBAD

- Making reaction with 1:5 ratio between number of inserts and backbone

|  |  |  |
| --- | --- | --- |
|  | Concentration | Amount Needed |
| CrtEBI-HQ | ng/ul | ul |
| pBAD | ng/ul | ul |
| 2xGibson mix | -------- | ul |

- RESULT:

# Week 39

Who worked: Simen, Alec, Prabin, Linnea

### Colony PCR

- PCR of colonies from last Gibson assembly, (week 38)

### Expression Test

- Expression test of cultures 4 and 5 from colony PCR

- 2% Arabinose, 4hrs

### Mini-Prep

- Made liquid cultures of colony 5 -> miniprep -> send for sequencing

### Colony PCR

- Colony PCR of pBAD-crtE, amplifying pBAD vector – want to make an empty pBAD vector as control

- RESULT: No pBAD amplification? No growth on grid plate?

## ------------------------------HQ-Part------------------------------

### Colony PCR

- PCR of colonies from last gibson assembly, week 38

### Expression test

- Expression test of colonies 1, 7, 8 and 11

- 2% arabinose, 4hrs

- NOTE: out of arabinose, only colonies 1 and 7 induced

- RESULT: For both 1 and 7, the induced cultures had a brighter red colour than the un-induced cultures

### Mini-Prep

- Made liquid cultures of colonies 1 and 7 --> mini prep

### Sequencing

- DNA from mini-prepped colonies send for sequencing

### Colony PCR

- for making pBAD control

- This is the same as what was done for our own sample, not from the HQ

# Week 40

Who worked: Simen, Prabin, Marthe, Kristin, Alec

### Transformation

- Transformation of cells with empty pBAD vector (to be used as control)

### Gibson Assembly

- Making reaction with 1:3 ratio between number of inserts and backbones

|  |  |  |
| --- | --- | --- |
|  | Concentration | Amount Needed |
| CrtEB | 108.4 ng/ul | 1 ul |
| CrtI | 100 ng/ul | 0.84 ul |
| 2xGibson mix | -------- | 1.84 ul |

- RESULT: 23 colonies on gel

### Colony PCR

- Colony PCR of colonies from transformation.

- RESULT: no colonies with crtEBI -> the Gibson was a failure

### Sequencing

- DNA from colonies 1, 5 and 7 sent for sequencing

### Gibson Assembly

- Repeating gibson assembly

###

### PCR

- PCR of colony 5. pBAD and crtEB as control

### Transformation result

- looking at result from transformation

### Mini-Prep

- Mini-prep of colony 5

- RESULT: concentration -- > colony 5 (169.9ng/ul)

### Freezer cultures

- Made freezer cultures of samples with empty pBAD vector

### Preculture

- Preculture of colony 5 and pBAD(empty vector), for expression test

### Expression test

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| t | Colony 5 (A600) | Colony 5 (A450) | pBAD (A600) | pBAD (A450) |
| 0 | 0.029 | ---- | 0.029 |  |
| 30 | 0.036 | ---- | 0.034 |  |
| 60 | 0.045 | ---- | 0.052 |  |
| 90 | 0.075 | ---- | 0.048 |  |
| 120 | 0.134 | ---- | 0.181 |  |
| 150 | 0.186 | ---- | 0.228 |  |
| 180 | 0.305 | ---- | 0.387 |  |
| 195 | 0.377 | 0.532 | 0.445 | 0.605 |
| 210 | 0.479 | 0.644 | 0.472 | 0.624 |
| 225 | 0.453 | 0.603 | 0.539 | 0.705 |
| 240 | 0.504 | 0.695 | 0.59 | 0.788 |
| 255 | 0.513 | 0.785 | 0.66 | 0.858 |
| 270 | 0.670 | 0.884 | 0.727 | 0.932 |
| 285 | 0.765 | 0.956 | 0.793 | 0.983 |
| 300 | 0.842 | 1.036 | 0.841 | 1.027 |
| 315 | 0.892 | 1.083 | 0.882 | 1.062 |
| 330 | 0.97 | 1.14 | 0.901 | 1.089 |
| 345 | 1.019 | 1.187 | 0.939 | 1.118 |
| 360 | 1.064 | 1.227 | 0.96 | 1.137 |
| 375 | 1.091 | 1.258 | 0.974 | 1.158 |
| 390 | 1.111 | 1.282 | 0.979 | 1.167 |
| 405 | 1.136 | 1.301 | 0.994 | 1.178 |

## ------------------------------HQ-Part------------------------------

### Mini-Prep

- Mini-prep of colonies 1 and 7

- RESULT: concentration -- > colony 1 (212.7ng/ul), colony 7 (139.5ng/ul)

### Freezer cultures

- Made freezer cultures of colonies 1, 7, 8 (original HQ sample)

### Preculture

- Preculture of colony 1 and 8, for expression test

### Expression test

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| t | Colony 1 (A600) | Colony 1 (A450) | Colony 8 (A600) | Colony 8 (A450) |
| 0 | 0.028 | ---- | 0.016 | ---- |
| 30 | 0.033 | ---- | 0.016 | ---- |
| 60 | 0.042 | ---- | 0.027 | ---- |
| 90 | 0.079 | ---- | 0.056 | ---- |
| 120 | 0.937 | ---- | 0.108 | ---- |
| 150 | 0.175 | ---- | 0.142 | ---- |
| 180 | 0.297 | ---- | 0.244 | ---- |
| 195 | 0.375 | 0518 | 0.306 | 0.436 |
| 210 | 0.474 | 0.626 | 0.397 | 0.540 |
| 225 | 0.440 | 0.599 | 0.503 | 0.650 |
| 240 | 0.490 | 0.674 | 0.477 | 0.658 |
| 255 | 0.560 | 0.755 | 0.553 | 0.735 |
| 270 | 0.642 | 0.843 | 0.620 | 0.826 |
| 285 | 0.720 | 0.917 | 0.705 | 0.895 |
| 300 | 0.784 | 0.982 | 0.766 | 0.968 |
| 315 | 0.826 | 1.026 | 0.833 | 1.016 |
| 330 | 0.888 | 1.076 | 0.879 | 1.066 |
| 345 | 0.920 | 1.111 | 0.922 | 1.105 |
| 360 | 0.952 | 1.136 | 0.947 | 1.129 |
| 375 | 0.972 | 1.158 | 0.962 | 1.151 |
| 390 | 0.986 | 1.176 | 0.985 | 1.171 |
| 405 | 1.005 | 1.188 | 0.994 | 1.185 |

# Week 41

Who worked: Simen, Alec, Kristin, Marthe, Prabin, Bianca

### Colony PCR

- Colony PCR for crtEBI

- RESULT: None of the colonies have the insert

### Preculture

- Preculture of crtEBI construct in DH5a and BL21 cells, the HQ-part (Bba\_274100) in BL21 cells, BL21 cells with the pBAD vector (no insert)

### Expression culture

- Expression culture of precultures

- 16hrs, 2% arabinose

- RESULT: Successful recovery of lycopene containing cells from the HQ-part (Bba\_K274100), our cells failed to produce lycopene

### Sequencing

- Sequencing of our crtEBI construct

### Solar Cell Prototyping

- Made a prototype solar cell from the induced cultures of BL21 cells with the HQ part (Bba\_K274100) in the pBAD vector

- RESULT: Titanium dioxide layer cracked and flaked, very poor voltage produced.

# Week 42

Who worked: Simen, Alec, Prabin

### Preculture

- Preculture of crtEBI and pBAD vector (no insert) in DH5a cells and in BL21 cells

### Expression test

- Expression culture of the 4 different precultures

- Incubation for 40hrs, 2% arabinose

- RESULT: Successful recovery of lycopene containing cells. Found out our construct produces more lycopene in DH5a than in BL21

### SDS-PAGE

- SDS-PAGE of last expression culture, to ensure that crtE, I and B are all expressed when appropriate

- RESULT: Gel was inconclusive but suggests only small levels of CrtE, CrtB, and CrtI expression.

### Lycopene extraction

- Lycopene extraction of the cultures above

- RESULT: Our construct, crtEBI, produces measurable amounts of lycopene, but not as much of the HQ part we ordered for characterization.

### Solar Cell Prototyping

- Made a prototype solar cell from the induced cultures of BL21 cells with the pBAD vector (no insert), our EBI construct in DH5a cells, BL21 cells with the HQ part (Bba\_K274100) in the pBAD vector

- RESULT: Final results were promising: 20mV more was produced in light conditions than in dark conditions when the panel was made with our crtEBI construct, whereas the difference was 40mV when the Bba\_K274100 cells were used in the cell. However, the pBAD cells flaked far too much to be usable in a cell, no amperage was detected and voltage decreased very slowly after removal of light source. It also produced voltage in natural light conditions (through a window on a cloudy day).

## Contact information

Address
:   Kristine Bonnevies hus, Universitetet i Oslo
:   Blindernveien 31, 0371 Oslo, Norway

Email
:   uioslonorway@gmail.com

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# Team:UiOslo Norway/Experiments

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UiOslo

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### Experimenting is fun!

Gel Extraction

### MATERIALS:

* Scalpel blade
* Thermo Scientific GeneJET gel extractionkit containing:

+ 100-100ul Binding Buffer
+ GeneJET Purificatin column
+ 700ul Wash Buffer (Pre-mixed with the appropriate amount of 99% ethanol)

* 1.5mL Eppendorf tube
* 51ul Reverse osmosis water

### EQUIPMENT:

* Thermocycler, heating block, or water bath
* Desktop Centrifuge
* Nanodrop spectrophotometer

### PROTOCOL:

- Excise the band of interest from the agarose gel

- Add 1μL Binding Buffer for every 1mg of agarose gel

- Incubate at 50°C – 60°C for 10 minutes, inverting every 2 minutes

(Incubate for longer and invert every minute if gel hasn't fully dissolved)

- Transfer gel solution to spin column

- Centrifuge at 12 000g for 1 minute, discard flowthrough

- Add wash buffer to the column

- Centrifuge a second time at 12 000g for 1 minute, discard flowthrough

- Centrifuge a third time at 12 000g for 1 minute, discard flowthrough

- Place the column into a new Eppendorf tube

- Add 50μL reverse osmosis water to the column

- Centrifuge a fourth time at 12 000g for 1 minute, discard flowthrough

- Initialize and blank Nanodrop spectrophotometer with 1μL reverse osmosis water

- Measure DNA concentration with Nanodrop spectrophotometer

- Store purified DNA at -20°C

REFERENCE: From Thermo Scientific GeneJET Gel Extraction Kit protocol

DNA Extraction from Bacterial Cells

### MATERIALS:

* 5mL LB media
* 5μL 100mg/mL ampicillin (Or an appropriate amount of another antibiotic)
* 50mL Falcon tube or similar container
* Agar plate with colonies of interest
* Tape
* QIAprep Spin Miniprep Kit containing:

+ 250μL Buffer P1
+ 250μL Buffer P2
+ 350μL Buffer N3
+ QIAprep Spin column
+ 0.75mL Buffer PE

* 1.5 mL Eppendorf tube
* 51μL Reverse osmosis water

### EQUIPMENT:

* Gas flame or biological materials hood
* Shaking Incubator
* Desktop Centrifuge
* Nanodrop spectrophotometer
* -20°C freezer

### PROTOCOL:

- Add ampicillin to LB media

- Inoculate media with the colony in the presence of a gas flame, biological materials hood or other sterile environment

- Incubate the culture for 16 hours at 37°C with 250 rpm shaking, ensure the container lid is not airtight such as by taping a loose lid on

- Centrifuge the culture for 10 minutes at 4000g and 4°C, discharge the supernatant

- Resuspend the culture in buffer P1 and transfer to 1.5 mL Eppendorf tube

- Add buffer P2, invert to mix

- Incubate for 4 minutes

- Add buffer N3 and invert to mix

- Centrifuge for 10 minutes at 12 000g and 4°C

- Pipette out the supernatant and apply to a QIAprep spin column

- Centrifuge for 1 minute at 12 000g and 4°C, discharge supernatant

- Add buffer PE

- Centrifuge a second time for 1 minute at 12 000g and 4°C, discharge supernatant

- Centrifuge a third time for 1 minute at 12 000g and 4°C, discharge supernatant

- Transfer column to an Eppendorf tube

- Add 50μL reverse osmosis water

- Centrifuge a fourth time for 1 minute at 12 000g and 4°C, discard column

- Initialize and blank Nanodrop spectrophotometer with 1μL reverse osmosis water

- Measure DNA concentration with Nanodrop spectrophotometer

- Store purified DNA at -20°C

Protocol from the QIAprep Spin Miniprep Kit

### MATERIALS:

* 2.40g Agarose
* 300mL TAE Buffer
* 5μL GelRed Dye

### EQUIPMENT:

* Microwave (~600W)
* Gel tray with holder
* Gel comb(s)

### PROTOCOL:

- Add agarose to TAE Buffer

- Microwave at full power for 1.5 minutes

- Swirl the solution briefly

- Microwave for another minute at full power

- Swirl briefly again

- Set up Gel tray in the holder

- Add GelRed to the tray

- Add ~50mL of agarose solution to the holder

- Store remain agarose solution at 59°C

- Mix agarose solution gently with a gel comb

- Place gel comb(s) in the appropriate slots in the gel tray

- Allow 20-30 minutes for the gel to set

- Use immediately

Agarose Gel Electrophoresis

### MATERIALS:

* 1μL 6x loading dye per sample
* 5μL of each sample
* TAE buffer
* 0.8% Agarose gel
* 3μL DNA ladder

### EQUIPMENT:

* Electrophoresis apparatus
* UV Gel reader w/ protective glass plate
* UV filtering safety glasses

### PROTOCOL:

- Add 1μL 6x loading dye to sample, pipette to mix. Repeat for all samples

- Fill electrophoresis apparatus with TAE buffer, insert gel

- Load DNA ladder to a well made by each comb

- Load 5μL sample into remaining wells

- Run apparatus for 30-40 minutes at a constant 90V

- Transfer gel to UV gel reader, cover with clear plastic

- Put on UV filtering safety glasses

- Read gel

SDS Gel Electrophoresis

### MATERIALS:

* 0.5-1mL Lysed cells (protein samples)
* 1.5 mL Eppendorf tubes
* 4x LDS loading buffer (volume equal to 1/3 total protein sample volume)
* 500μL 1x LDS loading buffer per sample
* Bolt 4-12% Bis-Tris Plus gel(s), 15 well
* ~500mL SDS buffer
* 5μL 1kDa Protein ladder per gel
* ~75-100mL Coomassie Blue staining solution
* ~150-200mL Destaining solution

### EQUIPMENT:

* Centrifuge
* Vortex machine
* Thermocycler or heating block
* Electrophoresis apparatus
* False back plate (if running an odd number of gels)
* Putty knife
* Shaking table
* Gel imager

### PROTOCOL:

- Spin down lysed cells at 12 000g and 4°C for 10 minutes

- Pipette out as much of the supernatant as possible, into new tubes

- Add 4x LDS loading buffer to supernatants in a 1:3 ratio

- Add 500μL 1x LDS loading buffer to each of the pellet samples

- Invert supernatant samples 3-4 times to mix

- Vortex pellet samples until dissolved into LDS buffer

- Heat shock samples at 95°C for 5 minutes

- Centrifuge samples for 10 minutes at 12 000g and 4°C

- Set up electrophoresis apparatus, using false back plate if not running a second gel in the same apparatus

- Remove Bolt gel from packaging, remove tape and comb from gel

- Place Bolt gel into electrophoresis apparatus facing the centre, secure the gel and back plate (if using) with provided mechanism

- Ensure the middle chamber is water-tight by adding a small amount of buffer to the chamber and checking for leaks, re-secure and re-test chamber if leaks are detected

- Fill the inner chamber until the buffer completely covers the gel wells

- Fill the outer chamber until the level of the buffer is 1cm above the exposed gel

- Load 5μL of protein ladder into a well of each gel

- Load 5μL of soluble sample into different wells, load only fully liquid portion, not gel portion

- Load 3μL of insoluble sample into different wells

- Close apparatus and start electrophoresis at a constant 220V for 35 minutes

- Remove gel from apparatus and remove plastic casing from gel using a putty knife

- Cut off the thick section of gel at the bottom of the gel

- Place gel in enough Coomassie blue solution to completely cover it

- Place container with gel and Coomassie blue solution onto shaking table at 120rpm for 1-2 hours

- Carefully decant Coomassie blue solution, add destaining solution until gel is completely covered

- Leave on shaking table at 120rpm for 1-2 hours

- Replace destaining solution, leave on shaking table for 16 hours

- Decant destaining solution and image with gel imager

Bacterial Precultures

### MATERIALS:

* 3mL LB media
* 3μL 100mg/mL ampicillin stock
* 1 15mL falcon tube
* Agar plate or freezer stock containing colonies of interest
* Colony picking tool
* Tape

### EQUIPMENT:

* Gas flame or biological materials hood
* Shaking incubator

### PROTOCOL:

- Add ampicillin stock to LB media in falcon tube

- Pick a colony or scratch the freezer culture with the picking tool while near a gas flame or in a biological materials hood

- Inoculate media with bacteria by dipping scraping into media and mixing briefly

- Leave cap unscrewed, secure with tape

- Place into shaking incubator at 37°C and 250 rpm for 16 hours

- Use within 24 hours of protocol completion

Bacterial Freezer Cultures

### MATERIALS:

* 10mL Bacterial preculture
* 150μL Glycerol
* 850μL LB media
* -80°C safe storage tubes

### EQUIPMENT:

* Centrifuge
* -80°C freezer

### PROTOCOL:

- Centrifuge bacterial preculture at 4 000g and 4°C for 10 minutes

- Gently decant and pipette out all LB media

- Add glycerol to LB media, shake vigorously to mix

- Resuspend cell pellet in 1mL 15% glycerol LB media

- Transfer to -80°C safe storage tube

- Transfer to -80°C freezer

Inducer Test Cultures

### MATERIALS:

* 5mL LB media per testing condition
* 5μL 100mg/mL Ampicillin stock testing condition
* 50μL Bacterial preculture testing condition
* Aluminum foil
* 50mL falcon tubes or similar incubation containers
* Tape
* 20% arabinose, filter sterilized (volume dependent on testing conditions)

### EQUIPMENT:

* Baffled Erlenmeyer flask
* Spectrophotometer
* Shaking incubator
* Centrifuge

### PROTOCOL:

- Add 5mL LB media per testing condition (plus 5mL for the control) to the baffled Erlenmeyer flask

- Add 5μL 100mg/mL ampicillin per testing condition (plus 5μL for the control) to the LB media

- Add 50μL bacterial preculture per testing condition (plus 50μL for the control) to LB+amp media

- Incubate at 37°C and 250rpm

- Check cell OD600 at 1.5 hours after inoculation and every 15 minutes thereafter until OD600 reaches ~0.4

- Transfer 5mL of LB+amp culture aliquots into 50mL falcon tubes or similar incubation containers, leaving lids unscrewed, securing with tape if necessary

- Separate cultures into different testing conditions and induce with arabinose

- Allow cells to incubate in the testing conditions

- Harvest cells by centrifugation at 4 000g and 4°C for 10 minutes

- Decant media and analyze immediately or store at -20°C

Making Chemically-competent cells

### MATERIALS:

* Agar plate of untransformed DH5α cells
* Colony picking tool
* 50mL LB media
* Aluminum foil
* 22.5mL Ice-cold 50mM CaCl2 solution
* Ice
* 0.25mL Glycerol
* 1.5mL Eppendorf tubes

### EQUIPMENT:

* 250mL baffled Erlenmeyer flask
* Gas flame or biological materials hood
* Shaking incubator
* Spectrophotometer

### PROTOCOL:

- Pick a colony from the agar plate and inoculate LB media in a 250mL baffled Erlenmeyer flask with the culture while in a biological materials hood or near a gas flame

- Cover the flask with aluminum foil

- Incubate culture at 37°C and shaking at 250rpm until the OD600 is between 0.35-0.4

- Centrifuge the culture for 10 minutes at 4 000g and 37°C

- Decant supernatant

- Resuspend cells in 20mL of CaCl2 solution

- Incubate for 20 minutes on ice

- Centrifuge the culture for 10 minutes at 4 000g and 37°C

- Decant supernatant

- Mix glycerol with 2.5mL of CaCl2

- Resuspend cells in CaCl2-glycerol solution

- Separate the cells into 100μL aliquots in the Eppendorf tubes

- Use competent cells immediately or store at -80°C

DH5α Cell Transformation

### MATERIALS:

* Ice
* 100μL aliquot of chemically-competent cells
* 1-10ng of DNA vector dissolved in 1-5μL MQ water
* 0.9mL S.O.C. Media
* LA+0.1mg/mL ampicillin plate

### EQUIPMENT:

* 42°C water bath
* Shaking incubator
* Non-shaking Incubator

### PROTOCOL:

- Thaw cells on ice if using frozen cells

- Add DNA solution to cell aliquot, tap gently to mix

- Incubate for 15 minutes on ice

- Heat shock cells in water bath for 45 seconds

- Incubate on ice for 2 minutes

- Add S.O.C. Media to cells, tap gently to mix

- Incubate cells at 37°C and 225rpm for 1 hour

- Plate 100μL of cells onto agar plate

- Incubate plate at 37°C for 16 hours

Taq PCR

### MATERIALS:

* 16.5μL MQ water for each reaction
* 2.5μL x10 ThermoPol buffer for each reaction
* 0.5μL each of 10mM Forward and 10mM Reverse primers for each reaction
* 0.5μL dNTPs (10mM each dNTP) for each reaction
* 0.125μL 5 000 U/mL Taq polymerase for each reaction
* PCR tube for each reaction
* 1μL DNA template (variable concentration) for each reaction

### EQUIPMENT:

* Thermocycler

### PROTOCOL:

- Mix a master mix of sufficient volume for all desired PCR runs. Each reaction consists of:

* MQ water
* ThermoPol buffer
* Forward primer
* Reverse primer
* dNTPs
* Taq polymerase

- Split master mix into 24μL aliquots in PCR tubes

- Add DNA template to each reaction

- Run the thermocycler with the following program:

* 95°C for 3 minutes
* 30 cycles of:
  + 95°C for 30 seconds
  + 50°C for 30 seconds
  + 68°C for 2.5 minutes
* 68°C for 5 minutes
* Hold at 12°C

Colony PCR & Grid Plates

### MATERIALS:

* LA+0.1mg/mL ampicillin plate
* 46.5μL MQ water per analyzed colony
* PCR tube per analyzed colony
* Colony pick tool per analyzed colony
* LA plate containing colonies
* 2.5μL x10 ThermoPol buffer per analyzed colony
* 0.5μL each of Forward and Reverse primers (10mM) per analyzed colony
* 0.5μL dNTPs (10mM each dNTP) per analyzed colony
* 0.125μL 5 000 U/mL Taq polymerase per analyzed colony
* 1μL x6 DNA loading buffer per analyzed colony
* TAE buffer
* 0.8% Agarose gel
* 3μL DNA ladder per gel comb

### EQUIPMENT:

* Marker
* Gas flame or biological materials hood
* Incubator
* Thermocycler
* Electrophoresis apparatus
* UV gel reader

### PROTOCOL:

- Use the marker to make a numbered grid on the LA+amp plate

- Add 30μL MQ water to a number of PCR tubes equal to the number of colonies to be tested

- Near a gas flame or in a biological materials hood:

* Use a pick tool to remove a colony from the LA plate
* Scratch a grid square with the pick tool
* Dip and spin the pick tool in a water-containing PCR tube
* Repeat for the desired number of colonies

- Place the grid plate into a 37°C incubator

- Boil the picked colonies at 102°C for 10 minutes then hold at 12°C

- Mix a master mix of sufficient volume for all desired PCR runs. Each reaction consists of:

* 16.5μL MQ water
* 2.5μL ThermoPol buffer
* 0.5μL Forward primer
* 0.5μL Reverse primer
* 0.5μL dNTPs
* 0.125μL Taq polymerase

- Split master mix into 24μL aliquots in PCR tubes

- Add 1μL of each the boiled colonies to separate master mix tubes

- Run the thermocycler with the following program:

* 95°C for 3 minutes
* 30 cycles of:
* + 95ºC for 30 seconds
  + 50ºC for 30 seconds
  + 68ºC for 2.5 minutes
  + 68ºC for 5 minutes
  + Hold at 12ºC

- Add 1μL 6x loading dye to 5μL of PCR product, pipette to mix. Repeat for all samples

- Fill electrophoresis apparatus with TAE buffer, insert gel

- Load 3μL DNA ladder to a well made by each comb

- Load 5μL sample into remaining wells

- Run apparatus for 30-40 minutes at a constant 90V

- Transfer gel to UV gel reader, cover with clear plastic

- Read gel

DpnI Digestion

### MATERIALS:

* 17μL Sample
* 2μL Cutsmart buffer x10
* 1μL 20 000 U/mL DpnI
* PCR tube or Eppendorf tube

### EQUIPMENT:

* Thermocycler, heating block or water bath

### PROTOCOL:

- To 17μL of sample, add 2μL x10 Cutsmart buffer and 1μL DpnI

- Mix gently by tapping

- Incubate at 37°C by chosen heating apparatus for 1 hour

Growth Curve Expression Test

### MATERIALS:

* 50mL LB Media per testing condition plus 50mL
* 50μL 100mg/mL ampicillin stock per testing condition plus 50μL
* 500μL Bacterial precultures of cultures of interest per testing condition plus 500μL
* 500μL Bacterial preculture of appropriate negative control per testing condition plus 500μL
* Aluminum foil
* ~5mL 20% arabinose, filter sterilized per testing condition plus 5mL

### EQUIPMENT:

* Baffled flasks
* Shaking incubator
* Spectrophotometer

### PROTOCOL:

- Add LB media per testing condition (plus 50mL for the control) to a baffled flask

- Add 100mg/mL ampicillin per testing condition (plus 50μL for the control) to the baffled flask

- Add bacterial preculture per testing condition to LB+amp media and mix thoroughly

- Transfer 50mL of LB+amp culture aliquots into 250mL Erlenmeyer flasks, covering the opening with aluminum foil

- Incubate at 37°C and 250rpm

- Check cell OD600 and take a 1mL sample every 30 minutes after inoculation until OD600 reaches ~0.4 for each culture, store samples at -20°C

- Induce with arabinose to a final arabinose concentration of 2%, induce cultures as they reach OD600~0.4, not all at once

- Allow cells to incubate at 37°C and 250rpm

- Check cell OD600, and take a 1mL sample every 15 minutes until 4 hours after induction, store samples at -20°C

Expression Cultures

### MATERIALS:

* 200mL LB media
* 200μL 100mg/mL ampicillin stock
* 2mL Preculture
* Aluminum foil
* 22mL 20% Arabinose stock solution

### EQUIPMENT:

* 0.5L baffled flask
* Shaking incubator
* Spectrophotometer
* Centrifuge

### PROTOCOL:

- Add 100mg/mL ampicillin stock to LB media in the 0.5mL baffled flask

- Add preculture to the baffled flask, cover the opening with aluminum foil

- Incubate at 37°C and 250rpm shaking

- Check cell OD600 every 30 minutes after inoculation until OD600 reaches ~0.4

- Induce with 20% arabinose

- Allow cells to incubate at 37°C and 250rpm for 4 hours

- Harvest cells by centrifugation at 4 000g and 46°C for 10 minutes

- Decant supernatant

Cell Lysis by Sonication

### MATERIALS:

* 5-10mL Cell Samples
* 0.625mL 80mM PMSF stock
* 500mL 10mM Tris-HCl stock
* 500μL 10mg/mL Lysozyme stock
* Ice

### EQUIPMENT:

* Centrifuge
* Sonicator w/ microprobe

### PROTOCOL:

- Centrifuge cells samples for 10 minutes at 4°C and 4 000g

- Mix a lysis buffer of:

* 0.625mL PMSF stock
* 500mL Tris-HCl stock
* 500μL Lysozyme stock

- Decant supernatant from the cell samples

- Resuspend each cell sample in 2mL lysis buffer, store samples on ice

- Store remaining lysis buffer at -20°C

- Sonicate each sample while maintaining them on ice at 39% intensity, for 2 minutes with a 10 seconds on, 20 seconds off pulse setting

- Analyze samples immediately or store at -20°C

Gibson Assembly

### MATERIALS:

* 1.5g PEG 8000
* 3 ml 1M Tris-HCl, pH 8.0
* 150μl 2M MgCl2
* 0.24μm syringe filters
* 25μl 1M DTT
* 50μl 10mM dNTPs
* 50μl 50mM NAD+
* 1μl T5 exonuclease (NEB Cat. #M0363S – 10units/μl)
* 31.25μl Phusion High Fidelity DNA Polymerase (NEB Cat. #M0530S – 2 units/μl)
* 250μl Taq Ligase (NEB Cat. #M0208S – 40 units/μl)
* 437.75 μl MilliQ water
* 100ng of insert DNA
* The number of linearized vector molecules equal to 1/3 the number of DNA inserts

### EQUIPMENT:

* Tube rotator
* Thermocycler, heating block or heating bath

### PROTOCOL:

- Make isothermal start mix by mixing:

* 1.5g PEG 8000
* 3mL 1 M Tris-HCl, pH 8.0
* 150μl 2M MgCl2

- Put on tube rotator until PEG is in solution

- Filter sterilize 25μl 1M DTT and 405μl isothermal start mix

- Make a 2X Gibson assembly master mix by mixing:

* The filter sterilized 25μl 1M DTT
* The filter sterilized 405μl isothermal start mix
* 50μl 50mM NAD+
* 1μl T5 exonuclease (NEB Cat. #M0363S – 10units/μl)
* 31.25μl Phusion High Fidelity DNA Polymerase (NEB Cat. #M0530S – 2 units/μl)
* 250μl Taq Ligase (NEB Cat. #M0208S – 40 units/μl)
* 437.75 μl MilliQ water

- Mix by pipetting gently

- Make 100μl aliquots

- Store isothermal mix and 2x Gibson assembly master mix at -20°C

- Make the cloning mix by mixing:

* 100ng of insert DNA
* The number of linearized vector molecules equal to 1/3 the number of DNA inserts
* 2x Gibson cloning mix equal to the combined volume of DNA insert and linearized vector

- Incubate for 1 hour at 50°C

- Transform cells immediately or store cloning mix at -20°C

Solar Cell Assembly

### MATERIALS:

* 127mg I2
* 10mL Ethylene Glycol
* 83mg KI
* 1mL 0.5M Tryptophane solution
* 15mL Falcon tube
* 1mL Cell pellet expressing lycopene
* 5mg TiO2
* 6mL MQ water
* Two 3cm x 2.5cm ITO glass plates
* 5mL Ethanol
* 1 Lint-free cloth
* Tape

### EQUIPMENT:

* Shaking incubator
* Centrifuge
* Multimeter
* Non-shaking incubator (Optional)
* Graphite pencil
* 2 Small butterfly clips
* Chemical fume hood

### PROTOCOL:

- Dissolve I2 in Ethylene Glycol

- Dissolve KI in iodide-glycol solution, set aside

- Add tryptophan solution into falcon tube

- Add cell pellet to tryptophan and mix gently to suspend

- Add TiO2

- Shake for 2 hours at 37°C

- Centrifuge for 10 minutes at 1000rpm

- Wash pellet by:

* Adding 2mL MQ water, shaking to mix
* Centrifuge for 3 200 rpm for 15 minutes
* Decant water
* Repeat the above 3 steps 2 additional times

- Clean ITO plates with ethanol and lint-free cloth, allow to air dry

- Find the conductive side of the plates with the multimeter by measuring resistance

- Tape down the glass plates, conductive side up

- Add paste from above steps to one ITO plate and smear evenly

- Allow paste to dry, gently heating will speed the process

- Use graphite pencil to cover the second plate in a layer of graphite/graphene

- Remove tape

- Sandwich the plates together, conductive sides facing inward and having overhangs where the tape was

- Attach butterfly clips to the sides of the ITO glass plates, leaving the area previously covered with tape exposed

- Gently add iodide solution into the cell by pipetting onto the exposed overhang while in a chemical fume hood

- Gently loosen clips to allow iodide solution to flow throughout the interior of the cell

Lycopene Extraction

### MATERIALS:

* 50mL Cell culture(s)
* Glass pipettes
* 1mL Acetone per cell culture
* 1mL Reverse osmosis water per cell culture
* One 2mL Eppendorf tube per cell culture
* 96-Plate (for Plate reader)

### EQUIPMENT:

* Spectrophotometer
* Centrifuge
* Chemical fume hood
* Vortex machine
* Plate reader

### PROTOCOL:

- Measure the OD600 of the cell cultures

- Centrifuge the cell cultures at 4 700g and 4°C for 10 minutes

- Decant media

- In a chemical fume hood, add acetone to cell cultures, close the container holding
the cell culture.

- Vortex for 5 minutes

- Centrifuge samples again at 4 700g and 4°C for 10 minutes

- Add water to 2mL Eppendorf tubes

- In a chemical fume hood, use a glass pipette to transfer acetone supernatant directly into the water

- Shake briefly to mix

- Transfer the extracts to wells on the plate reader plate

- Measure the extract’s OD472

Thin Layer Chromatography (TLC)

### MATERIALS:

* Acetone (volume varies)
* ~35mL of each cell culture to be analyzed
* 0.9mL Benzene per cell culture
* 1 piece of thin layer cellulose paper

### EQUIPMENT:

* Glass chromatography chamber
* Spectrophotometer
* Centrifuge
* Vortex machine

### PROTOCOL:

- Fill the chromatography chamber with acetone (around 4 cm from the bottom), remember to have the lid on to avoid evaporation.

- Measure OD600 of the cell cultures, ensure that the remaining steps use the same number of cells from each culture

- Centrifuge the cell cultures at 4 700g and 4°C for 5 minutes

- Decant supernatant

- Resuspend the pellet in 10% acetone dissolved in benzene, vortex to resuspend

- Centrifuge the cell cultures at 4 700g and 4°C for 2 minutes

- Pipette 100μL of benzene solution on to a thin layer cellulose paper and let dry

- Place the paper in the chamber, be sure that the dried cell extract is not in the acetone solution

- Allow the chamber to rest until the acetone is 2/3 up the paper

- Remove the paper from the chamber and allow it to dry

## Contact information

Address
:   Kristine Bonnevies hus, Universitetet i Oslo
:   Blindernveien 31, 0371 Oslo, Norway

Email
:   uioslonorway@gmail.com

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# Team:UiOslo Norway/Results

[Home](/Team%3AUiOslo_Norway)

Project

[Description](/Team%3AUiOslo_Norway/Description)
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About us

[Team](/Team%3AUiOslo_Norway/Team)
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Parts

[Basic Parts](/Team%3AUiOslo_Norway/Basic_Part)
[Composite Parts](/Team%3AUiOslo_Norway/Composite_Part)
[Existing Parts](/Team%3AUiOslo_Norway/Existing_Part)

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[Integrated Human Practices](/Team%3AUiOslo_Norway/Human_Practices)
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☰

UiOslo

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### The results are in...

* [Project Results](#results)
* [Future Considerations](#future)

## Results

### Cloning and Transformation

We confirmed a successful expression of *crtE* and *crtI* (figure 1), followed by *crtEB* and finally *crtEBI*.
![](https://static.igem.org/mediawiki/2019/0/07/T--UiOslo_Norway--SDS.png)*Figure 1: SDS-page of our induced basic parts (crtE, crtB and crtI).*

Transformation and successful cloning of *crtEBI* was confirmed by visually inspecting induced cultures for colour change. Cells with our construct were off-white before we transformed them, but showed red-ish/pink color complexes after transformation and induction (figure 2). Sequencing of the red-colored pellet showed a point mutation (deletion) in the end of the *crtI* gene. This mutation causes a frameshift, leading to the stop codon not being read during transcription. This is probably the reason we cannot get as much color in our bacteria as we would expect: lycopene is produced, but without a stop codon it will have a C-terminal extension that is not intended. This might affect both the transcription and translation of the pigment, hence lower the efficiency of lycopene production.

![](https://static.igem.org/mediawiki/2019/4/4e/T--UiOslo_Norway--our%26pBad.jpg)*Figure 2: Shows the pellet of cells from left to right: empty pBAD construct, uninduced empty pBAD construct, our induced crtEBI construct, and our uninduced crtEBI construct cells (far right)*

### Solar Cell Assembly

![](https://static.igem.org/mediawiki/2019/c/c0/T--UiOslo_Norway--Solarcell.jpg)*Figure 3: The setup of our solar cell.*We assembled our solar cell following the protocol described on our [experiment page.](https://2019.igem.org/Team%3AUiOslo_Norway/Experiments) We tried the assembly multiple times but had limited success building the solar cell (figure 3) as it produced the slightly higher voltage in the light condition than in the dark. However, the reading observed was not consistent and needs more fine tuning. There are multiple sources of error, like the dryness of the paste, thickness of the different layers and the coating of graphite. Even though we have our suspicions of what to improve, we did not have time to improve it.

### Chromatography

We decided to do thin layer chromatography to show lycopene production. Lycopene is a very hydrophobic compound which travels easily with the mobile organic phase, (acetone) and should be found at the top of the mobile phase front. This is also what we observed after the experiment was done (figure 4), but due to varying success in the extraction of lycopene during preparation, we could not use this result to compare the different samples. Because of little time and difficulties we had with extraction, we could only use this to prove that we have produced lycopene.

![](https://static.igem.org/mediawiki/2019/1/1f/T--UiOslo_Norway--TLC.png)*Figure 4: TLC result in visible (left) and in UV (right). From left to right: induced BBa\_K274120, uninduced BBa\_K2971004, induced BBa\_K2971004.*

## Future considerations

In the future we want to expand our project, introducing genes to enable the production of other pigments, which will allow an increased absorption spectrum. In addition, we would also like to test our construct in other bacteria, such as *D. radiodurans* or even yeast, if possible. We consider that in order to make our society more sustainable, we need innovative solutions that can complement already existing solar cells. Our hope is to, over time, technologies such as biogenic solar cells will replace the non-eco friendly energy sources.

![]()

## Contact information

Address
:   Kristine Bonnevies hus, Universitetet i Oslo
:   Blindernveien 31, 0371 Oslo, Norway

Email
:   uioslonorway@gmail.com

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