iGEM 2017 — Team Kent

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:Kent/Notebook

* Project
  [+ Description](https://2017.igem.org/Team%3AKent/Description)
  [+ Results](https://2017.igem.org/Team%3AKent/Results)
  [+ Modelling](https://2017.igem.org/Team%3AKent/Model)* Parts
    [+ Basic Parts](https://2017.igem.org/Team%3AKent/Basic_Part)
  * Notebook
    [+ Logbook](https://2017.igem.org/Team%3AKent/Notebook)
    [+ Experiments](https://2017.igem.org/Team%3AKent/Experiments)
    [+ Future Ideas](https://2017.igem.org/Team%3AKent/Improve)
  * [# ![](https://static.igem.org/mediawiki/2017/thumb/4/4f/T--Kent--Kenttemplogo.png/667px-T--Kent--Kenttemplogo.png)](https://2017.igem.org/Team%3AKent)
  * Safety
    [+ Project Safety](https://2017.igem.org/Team%3AKent/Safety)
  * Team
    [+ Meet the Team](https://2017.igem.org/Team%3AKent/Team)
    [+ Contribution](https://2017.igem.org/Team%3AKent/Contribution)
    [+ Attribution](https://2017.igem.org/Team%3AKent/Attributions)
  * Human Practices
    [+ Integrated HP](https://2017.igem.org/Team%3AKent/HP/Silver)
    [+ Public Engagement](https://2017.igem.org/Team%3AKent/Engagement)
    [+ Interlab](https://2017.igem.org/Team%3AKent/InterLab)
    [+ Collaboration](https://2017.igem.org/Team%3AKent/Collaborations)

![](https://static.igem.org/mediawiki/2017/thumb/9/91/T--Kent--LogbookHeader.png/800px-T--Kent--LogbookHeader.png)
Logbook
![](https://static.igem.org/mediawiki/2017/thumb/1/13/Logbookheader2.png/385px-Logbookheader2.png)

Project Logbook

Week 1

* Overnight cultures of DH5α E.coli cells
* Prepared competent E.coli cells and tested them with the iGEM
  competent cell test kit + results
  + Images of patched plates (?)
  + Bar chart of competent cells
  + Competency of cells - numbers are: 10pg/μl: 5 and 8;
    50pg/μl: 35 and 5; 100pg/μl 39 and 104
* ![](https://static.igem.org/mediawiki/2017/6/64/T--Kent--LogbookF5.png)![](https://static.igem.org/mediawiki/2017/b/b7/T--Kent--LogbookF6.png)
* Prepared LB medium, SOC medium, SOB medium and autoclaved materials

Week 2

* Made glycerol stocks of competent DH5α E. Coli overnights (1:1) with 50% glycerol stock and
  store at -80 C (keep for years)
* DNA mini prep PSB1C3 (Qiagen)
* Restriction digest using ECO R1 from Promega
* Run Gel of digested samples to confirm presence of desired plasmid (compare fragment sizes)
* Concentration of our DNA is 90 ng/mL (Nanodrop?)

Chloramphenicol plates to test whether plasmid (containing antibiotic resistance gene) was
incorporated into bacteria
Before----------------------After

![](https://static.igem.org/mediawiki/2017/3/35/T--Kent--LogbookF7.png)![](https://static.igem.org/mediawiki/2017/a/a1/T--Kent--LogbookF8.png)

* Did head shots in the green room

Week 3

* Transformation of GFP into Backbone (BBa\_K125500 into
  PSB1A3) + grow overnight in LB medium![](https://static.igem.org/mediawiki/2017/f/f5/T--Kent--LogbookF9.png)
* DNA mini prep (Qiagen)
* Agar gel (1%) with 2 digestion enzymes (Eco R1 and Pst I)![](https://static.igem.org/mediawiki/2017/0/01/T--Kent--LogbookF10.png)
* Talked to Dr. Garrett about mammalian cell use
* Ordered 3 different Cas13a Plasmids containing different mutations in the two HEPN domains

Week 4

* Our Plasmids arrived (Yaas!)
* Nanodrop of Week 3 GFP transformation into PSB1A3 vectors (2 attempts, low but gels were ok)
  ![](https://static.igem.org/mediawiki/2017/0/01/T--Kent--LogbookP1.png)
* Resuspension of stabs and overnight growth of (all three) cultures in LB medium
* Digestion with Eco R1 of all 6 samples (each twice = 3x2)
  ![](https://static.igem.org/mediawiki/2017/b/b8/T--Kent--LogbookP2.png)
* 0.7% Agarose Gel to confirm presence of plasmid
* Talked to Dr. Ellis, Dr. Kad & Taylor about Gibson assembly and Primer design
* Designed primers in Genome Compiler

Week 5

* Order of IDT gBlocks and primers
* Transformation of pSB1C3 + GFP into DH5α cells
* Plated and O/N growth at 37°C
* Next day, inoculate LB overnight cultures
* Miniprep DNA

Week 6

* Order of gRNAs
* Digest (GFP +pSB1C3) DNA with ECO R1 and Pst1 to confirm presence of plasmid (Fig 1)
  + DNA marker ( 1kb DNA life plus)
  + Undigested plasmid (pSB1C3 containing GFP)
  + Digested with Eco RI
  + Digested with PstI
  + Digested with both, ECO RI and PstI
![](https://static.igem.org/mediawiki/2017/1/10/T--Kent--LogbookF1.png)

Week 7

Amplification of dCas13a failed – order of new primers

Week 8

Competent DH5α cell preparation

Week 9

Amplification of dcas13a

* DNA marker (1kb DNA life plus) -- Lane 1
* dCas13a (3477 bp) -- Lane 3
* dCas13a (3477 bp) -- Lane 4

![](https://static.igem.org/mediawiki/2017/7/7f/T--Kent--LogbookF4.png)

Week 10

* Resuspension of gRNA
* Gibson assembly of pSB1CR + GFP with dCas13a
* Transformation and plated, incubated at 37°C overnight
* Set up LB overnights, patch plate and mini prep
* Digestion of whole plasmid with EcoR1 and Pst1 to check for band sizes (Fig 3)

![](https://static.igem.org/mediawiki/2017/4/49/T--Kent--LogbookF2.png)- Amplification of plasmid (whole gBlock + Backbone) with screening primers

Week 11

Successful amplification gBlock1

* Screening gel, complete construct PCR amplified with screening primers whole construct ~6600 bp
  + DNA 1 Kb ladder -- Lane 1
  + pSB1C3 containing GFP and dCas13a -- Lane 3-5x

![](https://static.igem.org/mediawiki/2017/6/6a/T--Kent--LogbookF3.png)

Week 12

* PCR amplify gBlock2 (dCas13a) and 1% agarose gel to confirm – failed
* Design and order of new dCas13a primers

Week 13

Run 1% agarose gel of gBlock 2 PCR products and pcDNA 3.1 vector amplification

* Once bacterial growth has been achieved, 500μL of the overnight liquid
  culture needs to be added to 500μL of 50% glycerol in a 2mL tube where it
  should be gently mixed
* The glycerol stock should then be frozen at -80 o C
  + Successive freeze and thaw cycles will reduce the stocks shelf life

Week 14

Run 1% agarose gel of gBlock 2 PCR products and pcDNA 3.1 vector amplification

* Once bacterial growth has been achieved, 500μL of the overnight liquid
  culture needs to be added to 500μL of 50% glycerol in a 2mL tube where it
  should be gently mixed
* The glycerol stock should then be frozen at -80 o C
  + Successive freeze and thaw cycles will reduce the stocks shelf life

Week 15

Run 1% agarose gel of gBlock 2 PCR products and pcDNA 3.1 vector amplification

* Once bacterial growth has been achieved, 500μL of the overnight liquid
  culture needs to be added to 500μL of 50% glycerol in a 2mL tube where it
  should be gently mixed
* The glycerol stock should then be frozen at -80 o C
  + Successive freeze and thaw cycles will reduce the stocks shelf life

Week 16

Run 1% agarose gel of gBlock 2 PCR products and pcDNA 3.1 vector amplification

* Once bacterial growth has been achieved, 500μL of the overnight liquid
  culture needs to be added to 500μL of 50% glycerol in a 2mL tube where it
  should be gently mixed
* The glycerol stock should then be frozen at -80 o C
  + Successive freeze and thaw cycles will reduce the stocks shelf life

![](https://static.igem.org/mediawiki/2017/thumb/c/c6/T--Kent--ScrollV2.png/101px-T--Kent--ScrollV2.png)

[![](https://static.igem.org/mediawiki/2017/thumb/a/a7/T--Kent--avokiddo.png/800px-T--Kent--avokiddo.png)](http://avokiddo.com/)
[![](https://static.igem.org/mediawiki/2017/thumb/1/1e/T--Kent--clicktravel.png/799px-T--Kent--clicktravel.png)](http://www.clicktravel.com)

[![](https://static.igem.org/mediawiki/2017/thumb/4/4e/T--Kent--bbsrc.png/800px-T--Kent--bbsrc.png)](http://www.bbsrc.ac.uk/)
[![](https://static.igem.org/mediawiki/2017/3/36/T--Kent--snapgene.png)](http://www.snapgene.com/)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)


[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:Kent/Experiments

* Project
  [+ Description](https://2017.igem.org/Team%3AKent/Description)
  [+ Modelling](https://2017.igem.org/Team%3AKent/Model)
  [+ Results](https://2017.igem.org/Team%3AKent/Results)* Parts
    [+ Basic Parts](https://2017.igem.org/Team%3AKent/Basic_Part)
  * Notebook
    [+ Logbook](https://2017.igem.org/Team%3AKent/Notebook)
    [+ Experiments](https://2017.igem.org/Team%3AKent/Experiments)
    [+ Future Ideas](https://2017.igem.org/Team%3AKent/Improve)
  * [# ![](https://static.igem.org/mediawiki/2017/thumb/4/4f/T--Kent--Kenttemplogo.png/667px-T--Kent--Kenttemplogo.png)](https://2017.igem.org/Team%3AKent)
  * Safety
    [+ Project Safety](https://2017.igem.org/Team%3AKent/Safety)
  * Team
    [+ Meet the Team](https://2017.igem.org/Team%3AKent/Team)
    [+ Contribution](https://2017.igem.org/Team%3AKent/Contribution)
    [+ Attribution](https://2017.igem.org/Team%3AKent/Attributions)
  * Human Practices
    [+ Silver](https://2017.igem.org/Team%3AKent/HP/Silver)
    [+ Gold](https://2017.igem.org/Team%3AKent/HP/Gold_Integrated)
    [+ Public Engagement](https://2017.igem.org/Team%3AKent/Engagement)
    [+ Interlab](https://2017.igem.org/Team%3AKent/InterLab)
    [+ Collaboration](https://2017.igem.org/Team%3AKent/Collaborations)

![](https://static.igem.org/mediawiki/2017/0/06/T--Kent--protocol_icon.png)
Experiments & Protocols
![](https://static.igem.org/mediawiki/2017/3/3e/T--Kent--protocol_icon2.png)

Basic Protocols

Production of Lysogeny broth (LB)

For 1 litre of LB a mixture of 10g of sodium chloride, 10g peptone, 5g of yeast extract as well as 1
litre of distilled water in a glass bottle. We then used a magnetic spinner to help mix the powders
with the water, we avoided shaking the glass bottle as it would cause froth and waste some of the
LB.

When making the LB we also made another litre batch and added 15g of agar extract to be able to
grow bacteria on plates.

Production of SOB medium and magnesium stock

Bringing together 20g of tryptone, 5g of yeast extract, 0.584g of NaCl, 0.186g of KCl and mixing it
with 990ml of millipure water (using the magnetic mixer again) which was then put in to autoclave
to sterilise it, after it was taken out and let for it to cool down to below 60 o C.

10ml of 2M Mg 2+ stock was then added and then brought to 100ml with millipure water, 0.2mm
filter sterilize was then used

Production of SOC medium and glucose stock

Once again bring 20g of tryptone, 5g of yeast of extract, 0.584g of NaCl, 0.186g of KCL, and then
bring 970 ml with millipure water and use the magnetic mixer once again, this was also then put in
to autoclave.

10ml of 2M Mg 2+ stock and then bring it to 100ml with milllipure water, filter sterilize it with 0.2m
and then final add 20ml of 1M glucose stock.

Production of Glycerol stock

If you wish to store bacteria long term, you will need to create a Glycerol Stock after
inoculating an overnight liquid culture

* Once bacterial growth has been achieved, 500μL of the overnight liquid
  culture needs to be added to 500μL of 50% glycerol in a 2mL tube where it
  should be gently mixed
* The glycerol stock should then be frozen at -80 o C
  + Successive freeze and thaw cycles will reduce the stocks shelf life

Running Agarose Gel

After the cells have been miniprepped and the plasmid put through a restriction digest, the agarose gel can be run.

* Make up some agarose. This is done by taking 0.5g of agarose powder and putting it in a
  250ml sterile conical flask, with 50ml of TAE buffer, then microwaving it in small pulses (20
  seconds then swirling it around) until it is dissolved. Don’t overheat it or it will evaporate too
  much. Make up the evaporated volume to 50ml with distilled water.
* Add 1 vial of cybersafe (ask technical services for a tube of it and add all of it)
* Line the white sides of the tank with the agarose solution, to seal it and prevent leakage. Use
  a p1000 pipette set to 1ml. Let it dry (about 5 mins max)
* Then pour all the agarose/sybrsafe solution into the tank and put in the comb. Let it set and
  solidify (maximum 30 mins)
* When the gel has set, remove the comb from the tank (gently!) and then cover the whole
  tank with TAE buffer, so there’s at least half a centimetre of TAE covering the gel.
* Now, the samples need to be loaded. Load some DNA markers (ask technical services for a
  tube of this and load the whole tube) into well 1( left hand side) and then choose what you
  load into wells 2, 3, and 4 etc. (make sure you note what’s in each lane!)
* Load all of your digests into the wells 2,3, and 4.
* Plug into a power supply and put the cover on. Run for 40 mins to an hour at 80v. The amps
  don’t matter.
* Once the visible markers have reached the half way point of the tank, turn off the power
  supply and drain the TAE buffer form the tank. Remove the gel with a spatula and place in a
  UV imaging box. Take an image of the gel under UV light, save it onto a USB stick.

Overnights protocol

After a transformation has been run and plates have been streaked and patched, overnight cultures will need to be made:

* Add 10mL of LB broth (not agar) into as many autoclaved conical flasks as
  needed
* Add 10uL of Chloramphenicol into each conical flask as well
* Using a pipette tip, scrape up some of the cell colonies on the agar plates
  prepared beforehand and drop it into the conical flask
* Cover up the flask using aluminum foil
* Incubate the cultures at 37oC and 180 rpm

Protocol for transformation/ heat shock

This requires chemically competent cells to be made beforehand. These must be stored at -80
degrees Celsius:

* First, the competent cells and the plasmid intended for transformation must be thawed on
  ice. Additionally, some 1.5ml Eppendorf tubes should be chilled on ice, along with some
  pipette tips.
* 100ul of the chemically competent cells are then pipetted (using the chilled pipette tip) into
  a chilled Eppendorf tube.
* 5ul of DNA is the pipetted into the tube with a chilled tip. This tube is then stored on ice for
  30 minutes.
* The tube is then placed in a water bath at 42 degrees Celsius for precisely 90 seconds. After
  90 seconds is up, the tube is transferred back to ice for 2 minutes.
* 900ul SOC medium is then added into the tube (with a normal pipette tip, doesn’t need to
  be chilled) and then mix very gently by pipetting up and down inside the tube.
* The tube is then incubated at 37 degrees Celsius for 45 minutes. The tube should not be
  shaken at all at this point.
* 100ul of the transformation mix is then pipetted into the centre of a plate containing LB agar
  and the appropriate antibiotic (for example, for plasmid pSB1C3, Chlorophenicol should be
  used, and for pSB1A3, use Ampicillin). Use 1ul of antibiotic for each ml of agar.
* In sterile conditions (Bunsen burner, gloves cleaned with IMS (ALLOWED TO DRY)), spread
  the bacteria around the plate by keeping the lid as closed as possible and inserting the
  spreader then turning the plate around to spread the cells. Then immediately close and
  store upside down.
* The remained of the transformation mix is then spun at the highest possible speed for 2
  minutes. The resulting pellet is then resuspended in 100ul of the existing medium and plated
  onto the LB and antibiotic plate.
* Incubate in a 37-degree Celsius incubator for 16-18 hours.
* Any colonies that result from this should be plated on a patch plate.
* This is done by taking a plate of LB agar with the appropriate antibiotic and dividing it up
  into sections by drawing a grid on the bottom. These sections are numbered and then using
  a sterile pipette tip, the colonies are gently streaked in each section- 1 colony per section.

Transformation Guidelines (QuickChange Protocol)

Store all XL1-Blue supercompetent cells at -80 o C (prevents loss of efficiency) as they are
sensitive to the smallest of temperature variations, even transferring tubes from one freezer
to another will result in loss of efficiency

Storage Conditions:

* XL1-Blue supercompetent cells should be stored at the bottom of a -80 o C freezer
* They should be placed at -80 o C directly from dry ice shipping container

Aliquoting Cells* Keep the XL1-Blue supercompetent cells on ice at all times
* Essential that BD-Falcon polypropylene tubes are places on ice before cells are
  thawed
* Cells must be aliquoted directly into prechilled tubes (Use of 14-mL BD Falcon Polypropylene Round-Bottom Tubes)
* These tubes must be used (BD Biosciences Catalog #352059) for the transformation
  protocol
* The heat-pulse steps’ duration is critical and has been optimized for the thickness as
  well as shape of these tubes

Length of Heat Pulse* Optimal efficiencies observed when cells are heat pulsed for 45 seconds
* Heat pulsing for at least 45 seconds is recommended, allowing for slight variations
  in incubation length
* Efficiencies noted to decrease sharply when pulsed for <30 seconds or >45 seconds
* This defined window of highest efficiency for the XL1-Blue cells results from heat
  pulse in step 3 of transformation protocol

Preparing Agar Plates for Colour Screening* To the LB agar, add
  + 80 µg/ml of 5-bromo- 4-chloro- 3-indolyl- β-D-galactopyranoside (X-gal)
  + 20 mM isopropy-1- thio β-D galactopyranoside (IPTG)
  + Appropriate antibiotic
* These are all added to prepare the LB agar plates for blue-white colour screening
* Alternatively
  + 100 μl of 10 mM IPTG and 100 μl of 2% X-gal can be spread on LB agar
    plates 30 minutes prior to plating transformations
* The IPTG must be prepared in sterile dH2O
* The X-gal must be prepared in dimethylformamide (DMF)
* IPTG and X-gal MUST NOT be mixed before being pipetted onto the plates since the
  chemicals may precipitate.

![](https://static.igem.org/mediawiki/2017/thumb/b/bf/T--Kent--ExperimentsConnect.png/133px-T--Kent--ExperimentsConnect.png)

Interlab Protocols

Calibration of OD 600 Reference Point

LUDOX-S40 must be used as a single point reference with the aim to attain a
ratiometric conversion factor, which in turn will be used to transform absorbance
data into standard OD 600 measurement.

Standard 1 cm path length spectrophotometer readings are instrument dependent,
while plate readers possess a path length less than 1 cm and are volume dependent.

Therefore, in this situation, there are 2 key objectives:

* Ratiometric conversion to transform Abs 600 measurements into OD 600
  measurements
* Accounting for instrument differences

Before starting the protocol, path length correction must be switched off. This is
because scattering increases with longer wavelengths therefore adjustment is
confounded by scattering solutions such as dense cells. However, many plate
readers have automatic path length correction which is based on volume
adjustment that uses ratio of absorbance measurements at 900 + 950 nm.
LUDOX solution is only weakly scattering so will produce low absorbance values

Use same cuvettes, plates and volumes that are going to be used in cell based assays

Materials:

* 1 mL 100% LUDOX
* H 2 O
* 96 well plate (black with flat, transparent/clear bottom)

Method

* 100 µl of LUDOX should be added into wells A1, B1, C1 and D2 (or 1mL into
  cuvette)
* 100 µl of H 2 O should be added into wells A2, B2, C2 and D2
* Measure absorbance 600nm of all samples in all standard measurement modes in instrument
* Record data

Production of Fluorescein stock solution

* Spin down the Fluorescein stock tube and ensure the pellet is at the tubes' bottom
* Prepare 2x fluorescein stock solution (100 µM)
  + Resuspend Fluorescein in 1mL 1xPBS
  + Ensure Fluorescein is properly dissolved
    After the resuspension, pipette up and down and examine the solution in
    the tip (if particulates are visisble, continue to mix solution until they
    disappear)

* Dilute the 2x Fluorescein stock solution
  + With 1xPBS to make 1x fluorescein solution
  + With resulting concentration of fluorescein stock solution 50 µM
    (500 µL of 2x fluorescein in 500 µL 1x PBS to make 1 mL of 50 µM (1x)
    fluorescein solution)

Fluorescein Fluorescence Standard Curve

A dilution series of Fluorescein in 4 replicates must be prepared where the
fluorescence is measured in a 96 well plate in standard mode on a plate reader. A
standard curve will be generated of fluorescence of fluorescein concentration. This
will be used to correct cell based readings to an equivalent fluorescein
concentration, which will then be converted into a GFP concentration.

Materials

* Fluorescein
* 10mL 1xPBS (Phosphate Buffered Saline)
* 96 well plate (black with flat, transparent/clear bottom)

Method

Serial dilutions need to be performed across columns 1-11
Column 12 must contain PBS buffer only

The plate will initially be setup fluorescein stock in column 1 and equal volume of1xPBS in columns 2-12

* Add 100 µL of PBS into wells A2-A12, B2-B12, C2-C12 and D2-D12
* Add 200 µL of Fluorescein 1x stock solution into A1, B1, C1 and D1
* Transfer 100 µL of Fluorescein stock solution from A1 into A2
* Mix A2 by pipetting up and down 3x and transfer 100 µL into A3
  Repeat the process for A3 into A4, A4 into A5, etc. until A11
* Mix A11 by pipetting up and down 3x and transfer 100 µL into liquid waste
* Repeat dilution series for rows B, C and D
* Measure fluorescence of all samples in all standard measurement modes in
  instrument
* Record the data

Measurement notes

* The plates can now be measured in the plate reader
* Standard GFP settings must be used (same as those used when measuring the
  cells):
  + Excitation 485nm+ Emission 530/30+ Turn off path length correction
* Would be ideal to repeat measurements with different settings
  + Generates series of standard curves to choose from
* Use number of settings that affect sensitivity (gain and/or slit width)
  + Also consider orbital averaging, top/bottom optics

Cell Measurement Protocol

The calibration measurements should be performed before the measurements on the cells are performed. This allows that the measurement process is understood
and that the cell measurements are taken under the same conditions.

Materials

* Competent cells (E.coli strain DH5-alpha)
* LB (Luria Bertani) media
* Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH –
  working stock 25 ug/mL)
* 50 mL Falcon tube (covered in foil to block light)
* Incubator at 37oC
* 1.5mL Eppendorf tubes for sample storage
* Ice bucket
* Pipettes
* 96 well plate (black with flat, transparent/clear bottom)
\*\*\*\*\*\*\*\*\*\*\*\*??????

Calcium Chloride Competent Cells

Prior Preparation

* Autoclave 50mM Calcium Chloride and keep it cold at about 4 o C
* For the starter cultures
  + + Add a colony of E.coli DH5cells to 5mL of LB
    + Incubate at 37 o C overnight

Method:

+ Keep cells on ice at all times where possible
+ To 100mLs of LB, add 100uL of cells from the overnight culture
+ Let it grow at 37 o C and 250 rpm (until it reaches OD 600 ~0.6-0.8)
+ Place cells on ice immediately to cool them once the correct OD 600 has been
  reached
+ Centrifuge at max speed for 10 mins and 4 o C
+ Discard supernatant
+ Resuspend the pellet in 50% of the original volume with ice-cold 50mM CaCl 2; In a 5omL culture, add 25mL CaCl 2
+ Allow them to sit on ice for 30 mins
+ Centrifuge at max speed for 10 mins at 4 o C
+ Discard the supernatant

Preparation of Competent Cells for Storage

Materials

+ Cell Line
+ Sterile LB
+ 10mM sterile and chilled Calcium Chloride
+ Dry ice
+ Acetone

Method

+ Inoculate the cells (either 1:50 or 1:100) into 50mL of LB
+ Grow them at 37 o C until OD600 is around 0.4-0.5
+ Place on ice for 10 minutes while Falcon tubes are pre-chilled
+ The cells should be harvested at 3000 rpm, 4C for 8 minutes
+ The pellet then needs to be resuspended in 1mL of 100mM CaCl 2 and 30%
  (v/v) glycerol
+ The resulting solution needs to be aliquoted into chilled Eppendorf tubes
  (100uL per tube)
+ Place each Eppendorf tube into an acetone dry ice bath to snap freeze them
+ Then store at -80 o C

![](https://static.igem.org/mediawiki/2017/thumb/b/bf/T--Kent--ExperimentsConnect.png/133px-T--Kent--ExperimentsConnect.png)

Complex Protocols

DNA Miniprep Kit (Qiagen)

Method: (passive + our)

* 2 x 5 mL of our ampicillin resistant bacteria, containing the plasmid of interest and grown
  overnight on LB medium, are centrifuged in falcon tubes at 4500 rpm for 6 minutes.
* The supernatant is removed and the pelleted bacteria are resuspended in 250μL of P1 buffer
  (containing 100 μg/mL RNase A). Thoroughly mix/ Vortex mix of the samples is required to
  ensure full resuspension. The samples are transferred into Eppendorf tubes.
* 250 μL of P2 buffer is added to each sample and gently mixed by inverting the tube ca. 10 times.
  This lysis reaction should not exceed 5 minutes.
* 350 μL of N3 buffer is pipetted to each sample, and gently but thoroughly mixed by inverting the
  tube ca. 10 times. The samples are then centrifuged in a table top centrifuge at 13.000 rpm for
  10 minutes.
* The supernatant contains our plasmid of interest, while the white pellet is cell debris. 800 μl of
  the supernatant are pipetted into Qiagen Spin Columns.
* The columns are centrifuged for 60 seconds. The plasmids are retained in a silica mesh, while
  remaining substances flow through the column into a collection tube.
* The column is washed with 500 μL of PB buffer and centrifuged (13.000 rpm for 60 sec) to
  remove any remaining nucleases which could interfere with further processing of the plasmids.
* 750 μL of PE buffer is added to each sample and centrifuged for 60 seconds to remove any
  remaining wash buffer. The flow through is discarded and the spin column is placed into a fresh
  Eppendorf tube.
* To elute the bound plasmid DNA, 50 μL of EB buffer is added to the column. After letting the
  samples stand for ca. 2 minutes, each tube is centrifuged at high speed (13.000 rpm) for 60
  seconds.
* The spin column is discarded, the Eppendorf tubes now contain our desired plasmid DNA.

Enzyme Digest Protocol

A restriction enzyme digestion is usually performed in a volume of 20μL with 0.2-1.5μg of substrate DNA and two-to tenfold excess of enzyme.

If a large volume of DNA or enzyme is used, abnormal results may occur

When pipetting the samples in the different lanes of the gel, the enzyme componentof the tube needs to make up 1μL.

Method:
1. The 5 lanes of the gel are as follows

* Marker
* Control (with no cutting enzyme)
* 1μL EcoR1
* 1μL Pst1
* 1μL EcoR1 and Pst1
2. Assemble the following components in a sterile tube:

![](https://static.igem.org/mediawiki/2017/thumb/8/8d/T--Kent--EnzymeDigest.png/800px-T--Kent--EnzymeDigest.png)

Note: Different lanes require different tubes to be made up
3. Mix the solution gently by pipetting up and down
4. Close the tube and centrifuge for a few seconds in a microcentrifuge
5. Incubate at the specific enzyme’s optimum temperature (37 o C in this case)for 1-4 hours
6. Add loading buffer to a 1 X final concentration and proceed to the gel analysis

PCR Protocol for Q5 High-Fidelity 2X Master Mix

All reaction components should be assembled on ice then quickly transferred to a thermocycler that’s been preheated to the denaturation temperature (98oC)

Components:
All the components should be mixed prior to use

![](https://static.igem.org/mediawiki/2017/c/cd/T--Kent--PCR1.png)

Method:

* Gently mix the reaction
* Collect all the liquid found at the bottom of the tube by a quick spin if needed
* Overlay the sample with mineral oil when using a PCR machine that doesn’t have a heated lid
* Transfer the PCR tubes to the PCR machine to begin thermocycling

Thermocycling conditions:

![](https://static.igem.org/mediawiki/2017/9/91/T--Kent--PCR2.png)

Annealing temperatures shouldn’t exceed 72 o C. You can use the NEB T m Calculator
found on the New England BioLabs website to calculate temperatures needed and
timings.

Guidelines
Template

* A high quality, purified DNA template is preferred as it greatly improves PCR success. Recommended amounts of such a template are shown below for a 50uL reaction:

![](https://static.igem.org/mediawiki/2017/thumb/f/f3/T--Kent--PCR3.png/800px-T--Kent--PCR3.png)

Primers

* Oligonucleotide primers should generally be 20-40 nucleotides long while having a GC content of 40-60%
* Best results are seen when using each primer at a final concentration of 0.5uM in the reaction

Mg2+ and additives

* The Q5 High-Fidelity Master Mix contains 2mM Mg++ when used at a 1X concentration, which is optimal for most PCR products

Deoxynucleotides

* Final concentration of dNTPs is 200uM of each deoxynucleotide in the 1X final concentration
* Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and isn’t recommended for use with uracil-containing primers or templates

Q5 High-Fidelity DNA Polymerase concentration

* Concentration in the Master Mix has been optimized for best results under a wide conditions range

Denaturation

* Initial denaturation of 30 seconds occurs at 98oC, which is enough for most amplicons from pure DNA templates.
* Though longer denaturation times going up to 3 minutes can be used for templates that require it

Annealing

* Optimal annealing temperatures for this Master Mix tend to be higher than for other PCR polymerases
* Typically 10-30 second annealing steps should be used at 3oC above the Tm of the lower Tm primer
* Temperature gradients can also be used to optimize the annealing temperature for each primer pair
  + For higher Tm primer pairs, two-step cycling without a separate annealing step can be used

Extension

* Recommended extension temperature is 72oC
  + With the recommended time being between 20-30 seconds per kb for complex, genomic samples.* * The time can be reduced to 10 seconds per kb for simpler templates (plasmid, E.coli, etc.) or complex templates smaller than 1kb
    * The extension time can be increased to 40 seconds per kb for cDNA or other long, complex templates if needed
    * A final extension of 2 minutes at 72oC is recommended

Cycle Number

* 25-35 cycles yield sufficient products generally
* For genomic amplicons, 30-35 cycles are advised

2-step PCR

* Used when primers have annealing temperatures exceeding or are equal to 72oC (≥ 72°C).
* This 2-step thermocycling protocol combines annealing and extension into one step

Amplification of long products

* When amplifying products > 6kb, you can increase the extension time to 40-50 seconds per kb.

PCR Product

* Products generated using this Master Mix have blunt ends
* If clonding is the next step then blunt-end cloning isn’t recommended
* If T/A-cloning is to be done, the DNA should be purified prior to A-addition, since the Q5 High-Fidelity DNA Polymerase will degrade any overhangs generated

![](https://static.igem.org/mediawiki/2017/thumb/c/c6/T--Kent--ScrollV2.png/101px-T--Kent--ScrollV2.png)

[![](https://static.igem.org/mediawiki/2017/thumb/a/a7/T--Kent--avokiddo.png/800px-T--Kent--avokiddo.png)](http://avokiddo.com/)
[![](https://static.igem.org/mediawiki/2017/thumb/1/1e/T--Kent--clicktravel.png/799px-T--Kent--clicktravel.png)](http://www.clicktravel.com)

[![](https://static.igem.org/mediawiki/2017/thumb/4/4e/T--Kent--bbsrc.png/800px-T--Kent--bbsrc.png)](http://www.bbsrc.ac.uk/)
[![](https://static.igem.org/mediawiki/2017/3/36/T--Kent--snapgene.png)](http://www.snapgene.com/)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:Kent/Results

* Project
  [+ Description](https://2017.igem.org/Team%3AKent/Description)
  [+ Results](https://2017.igem.org/Team%3AKent/Results)
  [+ Modelling](https://2017.igem.org/Team%3AKent/Model)* Parts
    [+ Parts](https://2017.igem.org/Team%3AKent/Parts)
    [+ Basic Parts](https://2017.igem.org/Team%3AKent/Basic_Part)
    [+ Composite Parts](https://2017.igem.org/Team%3AKent/Composite_Part)
    [+ Part Collection](https://2017.igem.org/Team%3AKent/Part_Collection)
  * Notebook
    [+ Logbook](https://2017.igem.org/Team%3AKent/Notebook)
    [+ Experiments](https://2017.igem.org/Team%3AKent/Experiments)
    [+ Future Ideas](https://2017.igem.org/Team%3AKent/Improve)
  * [# ![](https://static.igem.org/mediawiki/2017/thumb/4/4f/T--Kent--Kenttemplogo.png/667px-T--Kent--Kenttemplogo.png)](https://2017.igem.org/Team%3AKent)
  * Safety
    [+ Project Safety](https://2017.igem.org/Team%3AKent/Safety)
    [+ Hazard Signs](https://2017.igem.org/Team%3AKent/Signs)
  * Team
    [+ Meet the Team](https://2017.igem.org/Team%3AKent/Team)
    [+ Contribution](https://2017.igem.org/Team%3AKent/Contribution)
    [+ Attribution](https://2017.igem.org/Team%3AKent/Attributions)
  * Human Practices
    [+ Integrated Human Practices](https://2017.igem.org/Team%3AKent/HP/Silver)
    [+ Public Engagement](https://2017.igem.org/Team%3AKent/Engagement)
    [+ Interlab](https://2017.igem.org/Team%3AKent/InterLab)
    [+ Collaboration](https://2017.igem.org/Team%3AKent/Collaborations)

![](https://static.igem.org/mediawiki/2017/thumb/0/03/T--Kent--ResultsHeader.png/599px-T--Kent--ResultsHeader.png)
Results
![](https://static.igem.org/mediawiki/2017/thumb/2/2f/T--Kent--ResultsHeader2.png/800px-T--Kent--ResultsHeader2.png)

Our plan of creating our biobrick was pursued in two separate ways. First we attempted to clone the GFP with Standard 25 Prefix/Suffix from the biobrick library (BBa\_K6480131) as well as the dCas13a we obtained from Addgene (L. buccalis C2C2, addgene #834852). We also ordered the whole gene through Integrated DNA Technologies (IDT)

![](https://static.igem.org/mediawiki/2017/f/f8/T--Kent--R1.png)

Figure 1. 1% agarose gel electrophoresis diagnostic restriction digestion results of GFP with Standard 25 Prefix/Suffix (BBa\_K648013) in pSB1C3 . Lane 1: DNA Marker; Lane 2: undigested; lane 3; Single digestion with EcoRI; lane 4: single digestion with Pst1; lane 5: double digestion with EcoRI and PstI.

dCAS13a

![](https://static.igem.org/mediawiki/2017/1/1d/T--Kent--R2.png)
Figure 2: 1% agarose gel electrophoresis diagnostic restriction digestion results of L. buccalis C2C2, addgene #83485 in p2CT. Lane 1: DNA Marker; Lane 2: undigested; lane 3; Single digestion with EcoRI.

After successfully transforming these plasmids we designed primers for PCR amplification and Gibson Assembly3, adding a flexible linker sequence in between the GFP and the dCAS13a.

Because the dCAS13 gene contains three illegal restriction sites for iGEM submission (two EcoRI and one PstI) we also designed and ordered Quikchange4 PCR primers to remove these illegal sites.

After Gibson Assembly a screening PCR was performed with two screening primers, one 200 bp upstream and one 200bp downstream of the insert. Figure 3 shows a band the size approximately 4666 bp which indicates our insert (4266bp) plus an additional 400bp which account for the two screening primers on the 5’ and the 3’ end.

![](https://static.igem.org/mediawiki/2017/b/be/T--Kent--R3.png)
Figure 3. 1% agarose gel electrophoresis screening PCR results of dCAS13a - GFP in pSB1C3 . Lane 1: DNA Marker; Lane 2: negative control; lane 3; Screening PCR; lane 4: Screening PCR; lane 5: Screening PCR.

In order for our construct to work, it had to be transfected into HEK2935, a mammalian cell line. RNA localisation in bacteria is a contentious and poorly understood field. It does happen but not much is known about how, where and why6. In mammalian cells it is much better understood and poor localization has been linked with disease states7,8. Also because mammalian cells have cellular compartments, signals sequences can be added to the protein. We added nuclear localization sequences on both the N- and the C-terminal of the protein, so that unbound dCAS13a-GFP would localize in the nucleus until it is bound to an mRNA to track.

We transfected our construct (BBa\_K2340000) with a guide RNA plasmid (BBa\_K2340001 - BBa\_K2340011) (1:2 ratio) into HEK293 cells using Lipofectamine™ 2000 in a 24 well plate in media and incubated for 24 hours at 37℃. This was viewed under a brightfield microscope at 100x magnification.

![](https://static.igem.org/mediawiki/2017/8/81/T--Kent--R4.png)
Figure 4. Plate layout for transfection and brightfield microscopy. Well 1: Cas13 + Rab13 (1), well 2: Cas13 + Rab13 (2), well 3: Cas13 + Rab13 (3), well 4: Cas13 + EV, well 5: Rab13 (1) + EV, well 6: Pkp4 (1) + EV, well 7: Cas13 + Pkp4 (1), well 8: Cas13 + Pkp4 (2), well 9: Cas13 + Pkp4 (3), well 10: Inpp1 (1)+ EV, well 11: EV + ꞵ-actin (1), well 12: EV, well 13: Cas13 + Inpp1 (1), well 14: Cas13 + Inpp1 (2), well 15: Cas13 + Inpp1 (3), well 16: Transfection reagent control, well 17: parental, well 18: GFP control, well 19: Cas13 + ꞵ-actin (1), well 20: Cas13 + ꞵ-actin (2), well 21: Cas13 + ꞵ-actin (3). EV: Empty vector. Transfection reagent control: Lipofectamine™ 2000 GFP Control: eGFP in pcDNA3 (Addgene plasmid # 13031)

In Figure 5 we see the results from the brightfield microscopy. We saw no expression of our construct (Fig 5A) but the GFP control did express (Fig. 5B), indicating that there was transfection. The difference may lie in the type of GFP. This version of the construct had a wtGFP, which is natively expressed at lower temperatures (15-20 oC) and folds poorly at 37oC. We aim to redesign the GFP found on our construct to an eGFP (which is mutated to perform better at 37oC) to submit to the registry.

![](https://static.igem.org/mediawiki/2017/f/fb/T--Kent--R5.png)
Figure 5. Brightfield microscopy results. A: well 19: Cas13 + ꞵ-actin (1); no expression after 24 hours. After 30 and 48 hours there was still no expression (not shown here) B well 18: GFP Control; GFP expression after 24 hours.

Conclusion

We successfully build one biobrick by fusing together a dCas13a gene with a wtGFP gene from the biobrick library, adding a flexible linker and two nuclear localization sequences to improve stability and reduce background noise. The function of our construct has not been able to proof due to the poor folding of the wtGFP at 37oC and the short time frame. We continued to tweak the design, mainly substituting the wtGFP to an eGFP. Future experiments could test how the protein would react with single a NLS and longer/shorter or rigid linker sequences.

References

* 1Rose, J. (2011). GFP with Standard 25 Prefix/Suffix. [WWW Document]. URL http://parts.igem.org/Part:BBa\_K648013
* 2Addgene (2016). p2CT-His-MBP-Lbu\_C2c2\_R472A\_H477A\_R1048A\_H1053A. [WWW Document]. URL http://www.addgene.org/83485/
* 3New England Biolabs (2017). Gibson Assembly® Master Mix. [WWW Document]. URL https://www.neb.com/products/e2611-gibson-assembly-master-mix#Product%20Information
* 4Agilent genomics (2017). Quikchange. [WWW Document]. URL http://www.genomics.agilent.com/en/Site-Directed-Mutagenesis/QuikChange/?cid=AG-PT-175&tabId=AG-PR-1160
* 5Thomas P., Smart, T.G. (2005). HEK293 cell line: A vehicle for the expression of recombinant proteins. Journal of Pharmacological and Toxicological Methods, 51: 187-200.
* 6Avraam Buskila, A.., Kannaiah, S. & Amster-Choder, O. (2014). RNA localization in bacteria. RNA Biology, 11(8): 1051-1060.
* 7Pyke, C., et al. (1992). Localization of Messenger RNA for MT 72,000 and 92,000 Type IV Collagenases in Human Skin Cancers by in Situ Hybridization. Cancer Research, 52: 1336-1341.
* 8Bashirullah, A., Cooperstock, R.L., Lipshitz, H.D. (1998). RNA localization in development. Annu Rev Biochem, 67: 335-94.

[![](https://static.igem.org/mediawiki/2017/thumb/a/a3/T--Kent--UKCwhite.png/800px-T--Kent--UKCwhite.png)](https://www.kent.ac.uk/)
[![](https://static.igem.org/mediawiki/2017/thumb/a/a7/T--Kent--avokiddo.png/800px-T--Kent--avokiddo.png)](http://avokiddo.com/)

[![](https://static.igem.org/mediawiki/2017/thumb/1/1e/T--Kent--clicktravel.png/799px-T--Kent--clicktravel.png)](http://www.clicktravel.com)
[![](https://static.igem.org/mediawiki/2017/thumb/4/4e/T--Kent--bbsrc.png/800px-T--Kent--bbsrc.png)](http://www.bbsrc.ac.uk/)
[![](https://static.igem.org/mediawiki/2017/3/36/T--Kent--snapgene.png)](http://www.snapgene.com/)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)