iGEM 2018 — Team Thessaloniki

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# Team:Thessaloniki/Notebook

### Notebook

#### {{iGEMParseDate(selected.pos + 1) + ' of ' + selected.month }}

March 2018

We presented our project idea to our Primary Investigators and Professors
of our Faculties. During this time period we focused our brainstorming
sessions on the enrichment of our project, we enlisted the materials needed
for the laboratory work and we began to develop our experimental workflow.

June 2018

During our first weeks in the lab we received Safety and Security
training by various members of the labs we worked in. We were taught
proper lab practice, sterile techniques and physical biosecurity, along
with proper waste disposal and emergency procedures. We were instructed
on the function and usage of the various equipment in the lab like
Biosafety Cabinets, centrifuges and more.

* General Laboratory Procedures
* Equipment Use
* Emergency response procedures
* Prevention and control of laboratory contaminations
* Handling of hazardous reagents
* Disposal of Buffers and Chemicals

Setting up the Lab This week we focused on gathering the material and
equipment needed for our laboratory work. We prepared solutions, agar
plates, Bacterial Growth Medium and a small batch of DH5a competent cells.
We also resuspended the parts we needed from the iGEM 2018 distribution
kit.

July 2018

Wet Lab-Dry Lab meetings focused on Human Practices

This week we continued setting up the lab and gathering the materials
needed for our experiments. We, also, conducted the Interlaboratory
Measurement Study.

The pThSSe\_59, pThSSe\_60 plasmids from Addgene and our first gBlocks
arrived. We focused on cloning the Talesp1 and Talesp2 stabilized cassette
in backbones with different ORIs and prepared the constructs for Flow
Cytometry.

August 2018

We successfully removed the two illegal PStI sited inside the pTHSSe\_59 and
pTHSSe\_60 plasmids.

We got our first results for the TAL Effector Stabilization system.

We focused on the design of our CRISPRi stabilization system.

This week we successfully cloned our Riboswitch Constructs and got our
first Flow Cytometry data. We, also, successfully cloned our Theophylline
Riboswitch in the Tale Stabilised cassette.

After a constructive wet-dry meeting we decided to go a step further and
design a RNA level control system. Together with the dry lab we simulated
different topologies and chose the one better suited for our system.

September 2018

This Week we started working on our AND Gate Constructs.

Our Interlaboratory Measurement Collab has started. We prepared and
measured the samples to be sent to the other iGEM Teams.

The pAN-PTet-dCas9 plasmid arrived and we started working on our CRISRi
stabilization system. The AND Gate gBlocks arrived.

We received our sequencing results

We conducted the flow cytometry measurements for our constructs and
analyzed the results.

October 2018

Submit parts


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# Team:Thessaloniki/Experiments

# Protocols

# Protocols

### Frozen Competent Cells

#### Materials

* Plate of cells to be made competent
* LB Broth
* CaCl2 solution 0,1M
* 100% glycerol
* Ice

#### Method

1. Inoculate 1 colony from a freshly grown plate into 50ml broth
2. Incubate cells overnight at 37°C in a shaking incubator
   (200-250 rpm)
3. Transfer cells into 500 ml LB Broth in a 500ml E. flask
4. Incubate at 250-300 rpm and 37°C for approx. 2,5 hours
   (or OD = 0.5). Centrifuge at 5000 rpm and 4°C for 15 min
5. Remove supernatant and resuspend pelleted cells in 250 ml 0,1 M
   CaCl2 solution
6. Incubate on ice for 20 min
7. Centrifuge at 5000 rpm for 15 min
8. Remove supernatant and resuspend pelleted cells in 43 ml 0,1 M
   CaCl2 solution
9. Incubate on ice for 2-12 hours

   **Note:** Maximum Incubation at 24 hours. Efficiency of
   Transformation is gradually reduced afterwards
10. Add 7ml 100% glycerol, mix well
11. Aliquot cells into prechilled 15ml tubes
12. Quick freeze in liquid nitrogen
13. Store (-80°C)

### Fresh Competent Cells

#### Materials

* Plate of cells to be made competent
* LB Broth
* CaCl2 solution 0,1M
* Ice

#### Method

1. Inoculate 1 colony from a freshly grown plate into 50ml LB
   Broth in a 500ml E. Flask
2. Incubate at 250-300 rpm and 37°C for approx. 3 hours (or
   until OD600 is about 0,375)

   **Note:** Transformation Effeciency will decrease if
   cells are allowed to grow to a density greater than 0,4 OD
3. Centrifuge 3000 rpm and 4°C for 7 minutes. No brake.
4. Remove supernatant and drain the tubes by keeping them inverted
   on an absorbent paper for 1 minute
5. Resuspend pelleted cells in 10ml ice-cold CaCl2 solution by
   pipetting gently up and down. Make sure that cells are fully
   resuspended. Keep on ice for 5 min
6. Centrifuge 3000 rpm and 4°C for 7 minutes. No brake.
7. Remove supernatant and drain the tubes by keeping them inverted
   on an absorbent paper for 1 minute
8. Resuspend pelleted cells in 2ml ice-cold CaCl2 solution by
   pipetting gently up and down. Make sure that cells are fully
   resuspended. Keep on ice.

   **Note:** Increased transformation efficiency next 24h,
   decrease after the 24 hour mark.

### Chemical Transformation

#### Materials

* Chemically competent cells
* DNA to be transformed
* LB agar with antibiotics
* Ice

#### Method

1. Thaw 100 μl of competent cells on ice.

   **Note:** Thawing at room temp decreases efficiency
2. Add DNA:
   1. For transformation of ligated products add 8μl of the
      ligation reaction mix
   2. For transformation of known plasmids add approx. 10-100
      μg DNA
3. Incubate cells on ice for 30min.

   **Note:** Cutting this incub time short slightly
   decreases transformation efficiency.
4. Heat shock cells at 42°C for 90 sec.

   **Note:** Optimal heat shock duration depends on the
   strain and the method used to make it competent.
5. Incubate on ice for 5 minutes
6. Heat shock cells at 42°C for 90 sec
7. Incubate on ice for 5 minutes
8. Add 1ml of SOC or LB Broth.

   **Note:** Using SOC increases transformation efficiency.
9. Incubate cells for 1 hour at 37°C in a shaking incubator
   (200-250rpm).

   **Note:** Incubation without shaking will decrease
   transformation efficiency

   **Note:** If using LB Broth, longer incubation up to 2
   hours can increase transformation Efficiency
10. Centrifuge at 8000rpm for 3 minutes
11. Remove 920μl of supernatant
12. Resuspend pelleted cells in the remaining LB broth or SOC.
13. Plate resuspended cells onto LB agar plates containing the
    appropriate antibiotic.
14. Let the plates dry in the incubator for approx. 30min.
15. Flip upside down and incubate for 16-18 hours.

### Growing Overnight Cultures

#### Materials

* 5-10 ml LB broth
* 5-10 μl antibiotic
* culture tubes

#### Method

Overnight cultures are prepared under sterile conditions using a
Bunsen burner

1. Add 5-10 ml ofLB media into a culture tubes.
2. Add 5-10 μl of appropriate antibiotic into the broth.
3. Using a loop, pick a single colony and inoculate the culture by
   dipping the loop into the LB broth.
4. Incubate overnight at 37°C shaking at 200-250 rpm.

### LB Broth

#### Materials

* 20 g LB broth (powder)
* 1 Litre Purified Water

#### Method

1. Add 20g LB broth to 1 litre purified water
2. Autoclave

### LB Agar

#### Materials

* 35 g LB Agar (powder)
* 1 Litre Purified Water

#### Method

1. Add 37g LB Agar to 1 litre purified water
2. Autoclave

### SOC Medium

We followed the [Quiagen
protocol](https://www.qiagen.com/se/resources/faq?id=d3be05d0-ec02-4ced-aabe-dcd2a1061920&lang=en&Print=1)

#### Materials

* 0.5% Yeast Extract
* 2% Tryptone
* 10 mM NaCl
* 2.5 mM KCl
* 10 mM MgCl2
* 10 mM MgSO4
* 20 mM Glucose\*

#### Notes

* Add Glucose after autoclaving the solution with the remaining
  ingredients, and letting it cool down. Sterilize the final
  solution by passing it through a 0.2 µm filter.
* SOC medium can be stored at room temperature and is stable for
  several years.

### Glycerol Stocks

#### Materials

* 500µl glycerol (50%)
* 500µl overnight culture in LB

#### Method

1. Add 500µl glycerol (80%) to 1.5ml eppendorf tube
2. Add 500µl overnight culture in LB
3. Store at -80°C

### PCR From Plasmid DNA Template

We followed the NEB PCR with hiFi Q5 master mix [Protocol](https://international.neb.com/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492)
for our PCR reactions.

#### Materials

* 2x Q5 High-Fidelity Master Mix
* 10μΜ Forward Primer
* 10μΜ Reverse Primer
* Template DNA
* Nuclease Free Water

##### For a 20 µL reaction:

|  |  |
| --- | --- |
| Q5 High-Fidelity 2X Master Mix | 10 μl |
| 10 µM Forward Primer | 1 μl |
| 10 µM Reverse Primer | 1 μl |
| Template DNA | variable (1-5 ng) |
| Nuclease-Free Water | to 20 μl |

**Note:** Reactions are set on ice

#### Thermocycling

|  |  |  |
| --- | --- | --- |
| Initial Denaturation | 95°C | 2 minutes |
| 25-35 cycles | 95°C (Denaturation) | 0.5-1 minute |
| 42-65°C (Annealing) | 0.5-1 minute |
| 72°C (Extension) | 1 min/kb |
| Final Extension | 72°C | 5 minutes |
| Hold | 4–10°C | Indefinite |

**Note:** We used the [NEB
™ Calculator](https://tmcalculator.neb.com/#!/main) to determine the annealing temperature.

### Colony PCR

We followed the Promega GoTaq® DNA Polymeras [Protocol](https://worldwide.promega.com/-/media/files/resources/protocols/product-information-sheets/g/gotaq-dna-polymerase-m300.pdf?la=en)
for our Colony PCR reactions.

#### Materials

* 5X Green or Colorless GoTaq® Reaction Buffer1
* GoTaq® DNA Polymerase
* PCR Nucleotide Mix
* Forward Primer
* Reverse Primer
* Template DNA
* Nuclease Free Water

##### For a 50 μl PCR Reaction:

|  |  |
| --- | --- |
| 5X Green or Colorless GoTaq® Reaction Buffer1 | 10 μl |
| PCR Nucleotide Mix, 10mM each | 1 μl |
| Forward Primer | 1 μl |
| Reverse Primer | 1 μl |
| GoTaq® DNA Polymerase (5u/µl) | 0,25 μl |
| template DNA | variable (1-5 ng) |
| Nuclease-Free Water | to 50 μl |

#### Method

We performed 10μl Colony PCR Reactions

#### Thermocycling

|  |  |  |
| --- | --- | --- |
| Initial Denaturation | 98°C | 30 seconds |
| 25-30 cycles | 98°C (Denaturation) | 10 seconds |
| 50–72°C (Annealing) | 30 seconds |
| 72°C (Extension) | 30 seconds/kb |
| Final Extension | 72°C | 2 minutes |
| Hold | 4–10°C | Indefinite |

**Notes:** Perform PCR using your standard parameters

* Reactions are set on ice
* Reactions are placed in a thermal cycler that has been
  preheated to 95°C
* The thermal cycling protocol has an initial denaturation step
  where samples are heated at 95°C for 2 minutes to ensure that
  the target

### Agarose Gel Electrophoresis

#### Equipment

* Casting tray
* Well combs
* Voltage source
* Gel box
* UV light source

#### Reagents

* TAE
* Agarose
* Ethidium bromide (stock concentration of 10 mg/mL)

#### Method

##### For a standard 1% agarose gel

1. Measure 1 g of agarose and mix agarose powder with 100 mL 1xTAE
   in a microwavable flask
2. Microwave for 1-3 min until the agarose is completely dissolved
   and let agarose solution cool down to about 50 °C
3. Add ethidium bromide (EtBr) to a final concentration of
   approximately 0.2-0.5 μg/mL. EtBr binds to the DNA and allows
   you to visualize the DNA under ultraviolet (UV) light
4. Pour the agarose into a gel tray with the well comb in place
5. Place newly poured gel at 4 °C for 10-15 mins OR let sit at
   room temperature for 20-30 mins, until it has completely
   solidified
6. Add loading buffer to each of your DNA samples
7. Once solidified, place the agarose gel into the gel box
   (electrophoresis unit). Fill gel box with 1xTAE (or TBE) until
   the gel is covered
8. Carefully load a molecular weight ladder into the first lane of
   the gel and your samples into the additional wells of the gel
9. Run the gel at 80-150 V until the dye line is approximately
   75-80% of the way down the gel. A typical run time is about 40
   minutes-1.5 hours, depending on the gel concentration and
   voltage
10. Turn OFF power, disconnect the electrodes from the power
    source, and then carefully remove the gel from the gel box
11. Using any device that has UV light, visualize your DNA
    fragments. The fragments of DNA are usually referred to as
    ‘bands’ due to their appearance on the gel

### Restriction Digestion

* **Restriction Enzyme:** [NEB
  enzyme finder](https://enzymefinder.neb.com/#!#nebheader) used to determine the appropriate
  restriction enzymes
* **Reaction buffer:** [NEB
  double digest finder](https://nebcloner.neb.com/#!/redigest) used to determine an appropriate
  buffer and protocol for the restriction enzymes used

### Ligation

We followed the NEB Ligation [Protocol](https://international.neb.com/Protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202)
with T4 DNA Ligase.

#### Materials

* Microcentrifuge Tubes
* Ice
* T4 DNA Ligase Buffer (10x)
* T4 DNA Ligase
* Vector DNA
* Insert DNA
* Nuclease Free Water

#### Method

1. Set up the following reaction in a microcentrifuge tube on ice

   |  |  |
   | --- | --- |
   | **Component** | **20 μl Reaction** |
   | T4 DNA Ligase Buffer (10x) | 2 μl |
   | T4 DNA Ligase | 1 μl |
   | Vector DNA | 50 ng |
   | Insert DNA | ng calculated using the [NeBioCalculator](http://nebiocalculator.neb.com/#!/ligation) |
   | Nuclease Free Water | To 20 μl |
2. For cohesive (sticky) ends, incubate at 16°C overnight or room
   temperature for 10 minutes. For blunt ends or single base
   overhangs, incubate at 16°C overnight or room temperature for 2
   hours
3. Heat inactivate at 65°C for 10 minutes
4. Chill on ice and transform 8μl of the reaction into 100 μl
   competent cells
**Notes:**

* T4 DNA Ligase should be added last
* Ligation using a molar ratio of 1:3 vector to insert
  increases the reaction’s efficiency
* The T4 DNA Ligase Buffer should be thawed and resuspended
  at room temperature

### Golden Gate Assembly

We followed the Golden Gate Assembly [Protocol](https://international.neb.com/protocols/2015/03/04/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-mix-e1600)
for Using NEB Golden Gate Assembly Mix

#### Materials

* Destination Plasmid
* Insert/Inserts
* NEB Golden Gate Buffer (10x)
* NEB Golden Gate Assembly Mix
* Nuclease-free H2O

#### Method

1. Set up a 20 μl reaction according to the following table

   |  |  |
   | --- | --- |
   | **Reagent** | **Assembly Reaction** |
   | Destination Plasmid 75ng/μl | 1 μl |
   | Insert: * If Precloned * If in amplicon Form | 75-100 ng each plasmid 2:1 molar ratio insert:vector |
   | NEB Golden Gate Buffer (10x) | 2 μl |
   | NEB Golden Gate Assembly Mix | 1 μl |
   | Nuclease-free H2O | to 20 μl |

#### Assembly Protocol

|  |  |
| --- | --- |
| **Number Of Inserts** | **Assembly Protocol** |
| 1-4 inserts | 37°C for 60 minutes → 55°C for 5 minutes |
| 5-10 inserts | (37°C for 1 minute → 16°C for 1 minute)x30 → 55°C for 5 minutes |

For our plasmid isolation we used the Macherey Νagel NucleoSpin®
Plasmid isolation Kit, unless otherwise stated.
For PCR Purification and Gel Extraction we used the Macherey Nagel
NucleoSpin® Gel and PCR Clean-up Kit, unless otherwise stated.

### dCas9 Toxicity Measurements

All measurements shown regarding Abs600 were taken using plate
reader.

##### Day 1

Inoculate one single colony of DH5a E.coli cells containing the
dcas9 cassette into 1 ml LB+Kanamycin in

2‐ml eppendorf tubes

and
grown at 37 °C, **250 rpm** overnight in a MultitronPro shaker
incubator.

##### Day 2

The overnight cultures were diluted **1:360** into 1ml LB+ kanamycin in
**2‐ml eppendorf** tubes and doxycycline was added to the following
final concentration: **0, 0.2, 0.4, 0.6, 0.8, 1.2, 4, 8**.The cultures
were grown at 37 °C, **250 rpm** in a MultitronPro shaker incubator.
**After 6 hours** measurements for Abs600 were performed using plate
reader

### Flow Cytometry Sample Preparation

**We followed the protocol from Adam Mayer et al [1].**

All measurements shown were taken by cytometry of cells in mid-log
growth except when noted.

##### Day1

Glycerol stocks of strains containing the plasmids of interest were
streaked on LB + 1.5% Agar plates and grown overnight at 37 °C

##### Day2

Single colonies were inoculated into 1 ml LB + antibiotics in **2‐ml
eppendorf tubes** and grown at 37 °C, **250 rpm**
overnight in a MultitronPro shaker incubator

##### Day3

The overnight growths were diluted 1:200 into 1 ml LB + antibiotics
in **2‐ml eppendorf tubes** and grown at 37 °C, **250 rpm**.

After 2 hours the growths were diluted 1:500 into prewarmed LB +
antibiotics + inducer where necessary in **2‐ml eppendorf tubes**
and grown at 37 °C, **250 rpm** for 5 hours.

After growth, 20 μl of culture sample was diluted into 180 μl PBS +
200 μg/ml kanamycin to inhibit translation. The samples were stored
at 4°C for 1 hour and then measurements were performed using the
CyFlow Cube8 Sysmex Partec Flow Cytometer.

[1] Meyer, A. J., Segall-Shapiro, T. H., & Voigt, C. A. (2018).
Marionette: E. coli containing 12 highly-optimized small molecule
sensors. BioRxiv. Retrieved from
http://biorxiv.org/content/early/2018/04/10/285866.abstract

### Plate Reader Sample Preparation for our Interlaboratory Collaboration

We followed the iGEM 2018 InterLab Study [Protocol](https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf)

# Primers

# Primers

|  |  |  |
| --- | --- | --- |
| **Primers Name** | **Sequence** | **Usage** |
| VR | attaccgcctttgagtgagc | * Amplification of IDT gBlocks * Colony PCR |
| VF2 | tgccacctgacgtctaagaa | * Amplification of IDT gBlocks * Colony PCR |
| E Vector | belongs to the Metabrick Platform | * Vector Amplification for insertion of BsaI sites in the Prefix and Suffix |
| P Vector | belongs to the Metabrick Platform | * Vector Amplification for insertion of BsaI sites in the Prefix and Suffix |
| E Primer | belongs to the Metabrick Platform | * Part Amplification for insertion of BsaI sites in the Prefix |
| P Primer | belongs to the Metabrick Platform | * Part Amplification for insertion of BsaI sites in the Suffix |
| X Primer | belongs to the Metabrick Platform | * Part Amplification for insertion of BsaI sites in the Suffix |
| S Primer | belongs to the Metabrick Platform | * Part Amplification for insertion of BsaI sites in the Prefix |
| P.Rem 1 | aGGTCTCcgcagcaccctgctgcccttg | * Removal of PstI illegal Site from the Addgene Plasmids pTHSSe\_59 & pTHSSe\_60 (Insertion Point 50) |
| P.Rem 4 | aGGTCTCcgtgtctcgacataccaaattgagtcatgg | * Removal of PstI illegal Site from the Addgene Plasmids pTHSSe\_59 & pTHSSe\_60 (Insertion Point 50) |
| P.Rem 2 | GGTCTCgctgcgataaggctacgatgtgg | * Removal of PstI illegal Site from the Addgene Plasmids pTHSSe\_59 & pTHSSe\_60 (Insertion Point 802) |
| P.Rem 3 | GGTCTCgacacggactgctgcaactctttcgtag | * Removal of PstI illegal Site from the Addgene Plasmids pTHSSe\_59 & pTHSSe\_60 (Insertion Point 802) |
| In 1 | AGGTCTCAGGAActtcagccaaaaaacttaagacc | * Amplification of Test Device 1 and Test Device 2 from pTHSSe\_59 & pTHSSe\_60, used for our Interlaboratory Study |
| In 2 | AGGTCTCAACTgacatagcgtataaacgcaga | * Amplification of Test Device 1 and Test Device 2 from pTHSSe\_59 & pTHSSe\_60, used for our Interlaboratory Study |
| Ri 1 | AGGTCTCATTCCTGGCTGAAACCTATTATGACG | * Amplification of pTHSSe\_59 & pTHSSe\_60 vectors for our TALEsp1 and TALEsp2 stabilised Riboswitch Constructs. |
| Ri 2 | AGGTCTCACAGTCGCTATGTCTTCTACTAGTAGC | * Amplification of pTHSSe\_59 & pTHSSe\_60 vectors for our TALEsp1 and TALEsp2 stabilised Riboswitch Constructs. |
| DC1 | AGGTCTCAGGAAgctttactccaccgttgg | * Amplification of pAN-PTet-dCas9 plasmid to make the insert RFC10 compatible |
| DC2 | AGGTCTCAACTGcctcagataaaatatttgctcatgagc | * Amplification of pAN-PTet-dCas9 plasmid to make the insert RFC10 compatible |
| Vi1 | AGGTCTCATTCCGCTCCGTGTCTTCCTC | * Amplification of pTHSSe\_59 & pTHSSe\_60 vectors for insertion of a BsaI site in the prefix |
| AG1 | AGGTCTCAATGTCCAGATTAGATAAAAGTAAAGTG | * Amplification of the ANDgate Plasmid to replace the J23114 Promoter with a Talesp1 psp1w1 promoter for the TetR expression |
| AG2 | AGGTCTCATTCCGGCTGAACTCTAGAAGC | * Amplification of the ANDgate Plasmid to replace the J23114 Promoter with a Talesp1 psp1w1 promoter for the TetR expression |
| YG1 | aggtctcaggaaGAAGACACGGAGCTCG | * Amplification of pTHSSe\_59 psp1w1 to replace the J23114 Promoter with a Talesp1 psp1w1 promoter for the TetR expression |
| YG2 (30) | aggtctcaACATATGGTATTTCTCCTCtttAAtttaaaca | * Amplification of pTHSSe\_59 to replace the J23114 Promoter with a Talesp1 psp1w1 promoter for the TetR expression |
| YG2 (32) | aggtctcaACATCTAGTACTTTCCTGTGTGA | * Amplification of pTHSSe\_59 to replace the J23114 Promoter with a Talesp1 psp1w1 promoter for the TetR expression |
| IA1 | aggtctcactttTTAAACAAAATTATTTGTAGAGGCTGTTTCGtc | * Amplification of pTHSSe\_59 psp1w1 vector for insertion of BsaI sites in the Prefix |
| IA2 | aggtctcaTACTAGAGCCAGGCATCAAATAAAACG | * Amplification of pTHSSe\_59 psp1w1 vector for insertion of BsaI sites in the Suffix * Amplification of pSB1C3 and pTHSSe\_59 for the Insertion of constitutively expressed sgRNA cassette. |
| IG1 | aggtctcatcaaGGCTGTAAGAATCCTATAGGTTGAG | * Amplification of pSB1C3 and pTHSSe\_59 for the Insertion of constitutively expressed sgRNA cassette. |
| GG1 | aggtctcattgacagctagctcagtcctGTTTTAGAG | * Amplification of pRha sgRNA cassette in order to replace pRha with psp1w1 |
| GG2 | aggtctcaagtataTTATTTGTAGAGCTCATCCATGCCATG | * Amplification of pRha sgRNA cassette in order to replace pRha with psp1w1 |
| GK1 | aggtctctaaagaggagaaatactagATGAGCAAAG | * Amplification of B0034-sfGFP for the incorporation of BsaI restriction sites. |
| GK2 | aggtctcaagtaTTATTTGTAGAGCTCATCCATGC | * Amplification of B0034-sfGFP for the incorporation of BsaI restriction sites. |

# Clonning Strategy

# Cloning Strategy

#### TALE stabilized Promoters

#### TALEsp1 / TALEsp2 - Stabilized Promoters

TALEsp1-Pupsp1 stabilized promoter /TALEsp2-Pupsp2 stabilized
promoter

TALEsp1 / TALEsp2 - Stabilized Promoters

##### Steps

1. PCR amplification of psb1c3 vector with a set of
   standardized primers.

   These primers belong to the Metabrick Platform [1] and
   incorporate **BsaI** restriction sites to the
   biobrick
   prefix and suffix.
2. PCR amplification of **pTHSSe\_59** and **pTHSSe\_60**
   plasmid with E,P standardized primers.

   These primers belong to the Metabrick platform and
   incorporate **BsaI** restriction sites to the
   plasmid’s
   biobrick prefix and suffix.
3. Golden Gate assembly of the PCR amplified products.

   The amplified products after restriction digestion with
   bsaI restriction enzyme, acquire complementary sticky
   ends
   and can be ligated together forming the **TALEsp1-Pupsp1-pSB1C3**,
   **TALEsp2-Pupsp2-pSB1C3** plasmids.
4. Restriction digestion with **EcoRI** and **PstI**,
   enzymes that are unique to prefix and suffix.

   Since the assembled **Talesp1-Pupsp1** / **Talesp2-Pupsp2**
   constructs are **RFC[10] compatible**, they can be
   easily assembled with other vectors. This can be
   achieved
   by cutting the **Talesp1-Pupsp1-pSB1C3** vector and
   the
   receiver vectors with two biobrick enzymes one of which
   having it’s restriction site to prefix and the other to
   suffix.
5. Ligation between the digested pieces.

#### Psp1w1 promoter characterization device

The Psp1w1 promoter characterization device emerged from the
assembly of three individual parts: **Psp1w1**, **sfGFP
(without
starting codon)** and **amplified pSB1C3** vector.

##### Steps

1. PCR amplification of **pSB1C3** vector with a set of
   standardized primers.
2. Design and synthesis of the **Psp1w1** and **sfGFP
   (without
   starting codon)** with the appropriate flanking
   regions.
3. Golden Gate assembly between **Psp1w1**, **sfGFP
   (without starting codon)** and the **amplified
   pSB1C3**
   vector.

   This reaction forms the final plasmid **Psp1w1-pSB1C3**
   containing the Psp1w1 promoter characterization device.

#### Talesp1-Psp1w1 stabilized promoter

The following procedure has been followed in order to replace
the Pupsp1 stabilized promoter with the Psp1w1 stabilized
promoter .The expression levels of the sfGFP driven by the
Psp1w1 promoter are lower than the sfGFP expression levels
driven by the Pupsp1 promoter.

##### Steps

1. PCR amplification of **Psp1w1-pSB1C3** plasmid with **In1**,
   **In2** primers.

   These primers amplify the Psp1w1 promoter
   characterization device and incorporate **BsaI**
   sites flanking the amplified device.
2. PCR amplification of **TALEsp1-pSB1C3** vector with **Ri1**,
   **Ri2** primers.

   These primers amplify the TALEsp1 cassette together with
   the plasmid backbone and incorporate BsaI restriction
   sites at the external regions of the amplified
   sequence.

   Restriction digestion with BsaI restriction enzyme of
   the previously amplified sequences creates sticky
   complementary ends and thus they can be assembled
   together.
3. Restriction digestion with BsaI restriction enzyme of the
   previously amplified sequences creates sticky complementary
   ends and thus they can be assembled together.

   This reaction forms the final plasmid **TALEsp1-Psp1w1-pSB1C3**
   plasmid containing the Psp1w1 TALE stabilized promoter.

#### Riboswitches

The final plasmids for the characterization of the theophylline
riboswitches consist of three individual parts:
**A(Pupsp1 promoter fused with riboswitch), B(sfGFP marker)
and C.**
Each part’s sequence is flanked by BsaI recognition sites and
after digestion with BsaI restriction enzyme, part A has sticky
ends with part B and part C, while part B has sticky ends with
part A and part C.

##### Steps

1. PCR amplification of **psb1c3 vector** with a set of
   standardized primers.

   These primers amplify the plasmid backbone without the
   insert, incorporate BsaI sites at prefix and suffix,
   forming the **amplified pSB1C3 vector (part C)**.
2. Design and synthesis of the **part A**, **part B**
   fragments with the appropriate flanking regions.
3. Golden Gate assembly between **part A**, **part B**
   and the
   **amplified pSB1C3 Vector** (part C). This reaction
   forms the
   final plasmids **Ribo12.1-pSB1C3** and **RiboT27-pSB1C3**
   containing the riboswitch characterization device.

#### TALEsp1 stabilized promoters with translational control.

##### Steps

1. PCR amplification of **RiboT27-pSB1C3** plasmid with **In1**,
   **In2**
   primers.

   These primers amplify the riboswitch characterization
   device and incorporate **BsaI** sites at the
   external
   regions of the amplified device.
2. PCR amplification of **pTHSSe\_59** plasmid with **Ri1**,
   **Ri2** primers.

   These primers amplify the TALEsp1 cassette together
   with the plasmid backbone and incorporate **BsaI**
   restriction sites flanking the amplified sequence.

   Restriction digestion with **BsaI** restriction
   enzyme of the previously amplified sequences, creates
   sticky complementary ends and thus they can be
   assembled together.
3. Golden Gate assembly reaction between the amplified
   products from step 1 and step 2.

   This reaction forms the **TALEsp1-Pupsp1-RT27-pSB1C3**
   plasmid containing the theophylline inducible TALE
   stabilized promoter.

#### CRISPRi system

Constructs used to test dcas9/sgrna repression.

#### pRha-sgRNA Cassette

pRha-sgRNA Cassette test construct

##### Steps

1. PCR amplification of **pSB1C3** vector and **pTHSSe\_59**
   plasmid with a set of standardized primers.

   These primers belong to the Metabrick
   Platform,
   amplify the plasmid backbones without the
   insert and incorporate **BsaI** sites at prefix and
   suffix,
   forming the **amplified pSB1C3** and **amplified
   pTHSSe\_59**
   vectors.
2. Design and synthesis of part 1 **(sgRNA construct)** and
   part 2 **(sfGFP construct)**.

   Each part’s sequence is flanked by BsaI recognition
   sites and after digestion with BsaI restriction enzyme,
   part 1 has sticky ends with part 2 and the **amplified
   pSB1C3** or **amplified pTHSSe\_59** vector,
   while part 2 has
   sticky ends with part 1 and the amplified vectors.
3. Golden gate Assembly between part 1, part2 and one of the
   **amplified pSB1C3** or **amplified pTHSSe\_59**
   vector.

   Depending on the amplified vector we chose, this
   reaction leads to the formation of the
   **sgRNA-sfGFP-pSB1C3** or the **sgRNA-sfGFP-pTHSSe\_59**
   plasmid.

   We transformed DH5a E.coli competent cells with
   **sgRNA-sfGFP-pTHSSe\_59** vector along with **dCas9-pSB3K3**
   vector in order to test the response function for the
   repression of the **J23104** promoter by the
   sgRNA/dCas9
   complex. This procedure is thoroughly described at the
   characterization page.

#### CRISPRi stabilized promoter

After testing the response function of the CRISPRi system we
replaced the rhamnose inducible promoter that is located
upstream of the sgRNA cassette with the constitutive Psp1w1
promoter.

##### Steps

1. Digestion with biobrick enzymes of the Psp1w1 promoter
   characterization device and ligation with digested **pTHSSe\_59**
   vector.

   Since the Psp1w1 promoter characterization device is
   compatible with Biobrick **RFC[10]**, it can be
   easily
   assembled with other **RFC[10]** compatible vectors.
   This
   requires digestion of the characterization device and
   the receiver vector with two enzymes having their
   restriction sites on the prefix and suffix, followed by
   ligation.

   These reactions forms the final plasmid **psp1w1-pTHSSe\_59**
   containing the Psp1w1 promoter characterization device.
2. PCR amplification of the **psp1w1-pTHSSe\_59** plasmid
   with **IG1**, **IA2** primers.

   These primers amplify the psp1w1 promoter sequence
   together with the pTHSSe\_59 plasmid backbone and
   incorporate **BsaI** restriction sites flanking the
   amplified sequence.
3. PCR amplification of the **sgRNA-sfGFP-1c3** plasmid
   with **GG1**, **GG2** primers.

   These primers amplify the sgrna cassette along with the
   stabilized **J23104** promoter cassette and
   incorporate
   **BsaI** restriction sites at the external regions
   of the
   amplified sequence.
4. Golden gate assembly of the amplified products.

   After digestion with **BsaI** restriction enzyme,the
   amplified sequences acquire complementary sticky ends
   and thus they can be assembled together forming the
   **Psp1w1-CRISPRi-stabilized promoter-pTHSSe\_59**
   vector.
5. Restriction digestion with two enzymes that are unique to
   prefix and suffix.

   Since the assembled **CRISPRi-stabilized-Psp1w1**
   construct
   is compatible with Biobrick **RFC[10]**, it can be
   easily
   assembled with other vectors. This can be achieved by
   cutting the **Psp1w1-sgRNA-stabilized
   promoter-pTHSSe\_59**
   vector and the receiver vectors with two enzymes having
   their restriction sites on the prefix and
   suffix,followed by ligation.

#### dCas9-tetR expression cassette

The dcas9-tetR expression cassette is located at the
pAN-Ptet-dcas9 plasmid. This plasmid was obtained from Addgene
and is not compatible with **RFC[10]** biobrick standard,
since it
lacks prefix and suffix. Furthermore there is one recognition
site of the **EcoRI** enzyme in the coding sequence of the
dCas9.
Taking all the above into consideration, we decided to modify
this plasmid in order to make it compatible with the **RFC[10]**
biobrick standard.

##### Steps

1. PCR amplification of **TALEsp1-pSB1C3** plasmid with **Vi1**,
   **Ri2** primers.

   These primers amplify pSB1C3 along with prefix, suffix
   and incorporate **BsaI** restriction sites and the
   external regions of the amplified sequence.
2. PCR amplification of **pAN-Ptet-dCas9** plasmid with **DC1**,
   **DC2** primers.

   These primers amplify the **dCas9-tetR-lacI**
   expression cassette and add **BsaI** restriction
   sites flanking the amplified sequence.
3. Golden gate assembly of the amplified products.

   After digestion with BsaI restriction enzyme,the
   amplified sequences acquire complementary sticky ends
   and thus they can be assemble together forming the
   **pAN-Ptet-dCas9-prefsuf** plasmid.
4. PCR amplification of pAN-Ptet-dcas-prefsuf plasmid with **dcm1**
   and **dcm2** primers and sequential Golden Gate
   assembly.

   These primers amplify the **dcas9-tetR-lacI**
   expression
   cassette along with the vector backbone and incorporate
   **BsaI** sites at the amplified sequence. After
   digestion
   with **BsaI**, followed by ligation this sequence
   can be
   circularized forming the initial plasmid with a single
   mutation at the EcorI recognition site.

#### AND GATE

#### AND Gate-pSB4K5 & AND Gate-pSB3K3

The following procedure has been followed in order to
incorporate the AND Gate circuit at **pSB4K5** and **pSB3K3**
plasmids.
Then, we evaluated the circuit’s behaviour at these plasmids of
different copy number.

##### Steps

1. Design and synthesis of the **part A**, **part B**
   fragments with the appropriate flanking regions.
2. PCR amplification of **pSB1C3** vector with a set of
   standardized primers.

   These primers belong to the Metabrick Platform and
   incorporate **BsaI** restriction sites to the
   biobrick prefix and suffix.
3. Golden Gate assembly reaction between **part A**,**part
   B** and
   the amplified **pSB1C3** vector. This reaction forms
   the
   final plasmids **AND Gate-pSB1C3** containing the
   AND gate
   circuit.
4. Restriction digestion with **EcoRI** and **SpeI**
   (enzymes unique to prefix and suffix)

   Since the assembled AND gate construct is compatible
   with **Biobrick RFC[10]**, it can be easily
   assembled with
   other vectors. This can be achieved by cutting the **AND
   Gate-pSB1C3** plasmid and the receiver vectors
   with two
   enzymes having their restriction sites on the prefix
   and suffix. Following this approach we digested and
   assembled the AND gate circuit with **pSB4K5** and
   **pSB3K3**
   vector, constructing the **AND Gate-pSB4K5** and **AND
   Gate-pSB3K3 plasmids**.

[1] Damalas, S. (2017, June 9). The MetaBrick platform for DNA manipulation
and standardization. Bridging Synthetic Biology standards for optimized
interoperability. <https://doi.org/10.17605/OSF.IO/8DHMU>

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Thessaloniki/Results

# Result

# Result

### Overview

This year, we aimed to create a toolbox of stabilized promoter systems that
allows for constant gene expression, independent of gene/plasmid copy
number. In order to achieve that, we designed two different stabilization
systems, utilizing TAL Effectors and CRISPR interference. To allow for
control over gene expression, we introduced a theophylline riboswitch that,
when induced with different concentrations of theophylline, achieves
further control over stabilized gene expression at the desired level. This
page contains all the results we obtained, the conclusions we arrived at,
the debugging we performed, as well as our future plans.

All measurements were performed in biological replicates (n=3), as a chassis
we used DH5a E. coli cells, maintained at mid-log phase and as reporter, we
measured sfGFP fluorescence intensity (ex. 488nm).

All relevant protocols can be found at [our
protocols page](https://2018.igem.org/Team%3AThessaloniki/Experiments)

In order to investigate the function of **TALEsp1-Pupsp1 stabilized
promoter**
([BBa\_K2839000](http://parts.igem.org/Part%3ABBa_K2839000)) and **TALEsp2-Pupsp2
stabilized promoter** (
[BBa\_K2839014](http://parts.igem.org/Part%3ABBa_K2839014)
) we
measured their fluorescence, as well as the fluorescence of a **constitutive
promoter** across 3 plasmids with different Origins of Replication
(psc101,
p15A, pUC19-derived pMB1). For the sfGFP fluorescence intensity
measurements, flow cytometry was our primary measuring method, while a
plate reader was also used.

Figure 1. TALE1sp1 Pupsp1, TALEsp2 Pupsp2, non
stabilized constitutive pT7A1w1 promoter flow cytometry fluorescence
measurements at three different copy numbers. Error bars represent
standard deviation from three biological replicates.

Figure 2. TALE1sp1 Pupsp1, TALEsp2 Pupsp2, non
stabilized constitutive pT7A1w1 promoter Plate Reader fluorescence
measurements at three different copy numbers. Error bars represent
standard deviation from three biological replicates.

The results demonstrate that sfGFP expression level, under the control of
**TALEsp1-Pupsp1 stabilized promoter** () and **TALEsp2-Pupsp2
stabilized promoter** ([BBa\_K2839014](http://parts.igem.org/Part%3ABBa_K2839014)),
remains stable when expressed from
vectors with different copy number. Whereas, sfGFP expression driven from a
non stabilized constitutive promoter changes when different copy number
plasmids are used for its expression.
Flow cytometry and plate reader data both point towards the decoupling of
sfGFP expression from the plasmid copy number.
Unexpected behaviour was seemingly observed by the constitutive promoter in
the flow cytometry assay. A possible cause might be cross-sample
contamination.

### CRISPRi stabilized promoter

In order to investigate the function of the CRISPRi stabilized promoter
(BBa\_K2839016), we measured its fluorescence outut, as well as a
constitutive promoter’s fluorescence across 2 plasmids with different
Origins of Replication (psc101, pUC19-derived pMB1). dCas9 driven by the
PTet promoter (BBa\_R0040), is expressed from a pSB3K3 vector (p15A ori),
co-transformed in the cells measured.

In order to prevent any growth rate reduction effects due to the
over-expression of dCas9 and ensure that it is expressed at a high enough
level so as to allow a wide range of copy number independence, the Optical
Density at 600nm of cells expressing dCas9 at different inducer
(doxycycline) concentrations was plotted over the concentration used 6
hours after induction (Figure 3). The desired level of expression was
determined to be achieved between 0.8 and 1.2 ng/ml of inducer. These 2
concentrations are later used to induce the expression of dCas9 in all
experiments.

Figure 3. Cell growth inhibition caused by
different expression levels of dCas9, induced by different
concentrations of doxycycline.

From figure 3, it is clear that cells viability start to significantly
decline after adding doxycycline to a final concentration of 1.2 ng/ml.

### Different Copy number Measurements.

**The dcas9 cassette is located on a standard pSB3K3 vector (p15A ori)
and
it’s expression, driven by tet repressible promoter, is remained
constant
upon the addition of two different concentrations of doxycycline (0.8
ng/ml, 1.2 ng/ml).**
We inserted **CRISPRi stabilized J23104 promoter** ([BBa\_K2839016](http://parts.igem.org/Part%3ABBa_K2839016))
into plasmids
with different Origins of Replication (psc101, pUC19-derived pMB1). In
order to investigate their stabilization effect on sfGFP expression across
different copy numbers we measured fluorescence, using sfGFP as a marker.
For the sfGFP fluorescence intensity measurements, we conducted flow
cytometry analysis.

Figure 4. **CRISPRi stabilized promoter** and non stabilized
constitutive pT7A1w1 promoter flow cytometry fluorescence measurements
at two different copy numbers. Error bars represent standard deviation
from three biological replicates.

Stabilization of the [BBa\_J23104](http://parts.igem.org/Part%3ABBa_J23104)
Anderson promoter was successful, as seen in Figures 5 and 6.

![Diagram image](https://static.igem.org/mediawiki/2018/e/e0/T--Thessaloniki--diagram-6.png)

Figure 5. Time assays of CRISPRi stabilized promoter at different dCas9
induction levels and copy numbers. Flow cytometry measurements were
performed at 1.5, 3.5 and 7.5 hours post-induction with doxycycline.

From figure 4 it is clear that sfGFP expression level, under the control of
**CRISPRi stabilized J23104 promoter** ([BBa\_K2839016](http://parts.igem.org/Part%3ABBa_K2839016))
with dCas9 levels induced
by 0.8 or 1.2 ng/ml doxycycline, remains stable when expressed from
different vectors, whereas, without dCas9 expression, sfGFP expression
level changes when different copy number plasmids are used for its
expression.

Higher induction level of dCas9 results in lower stabilization Error and
lower expression. This shows that dCas9 expression partially determines the
repression strength, even at seemingly not saturated levels of expression.
Stabilization Error (E) for 0.8ng/ml Doxycycline induction was calculated
to be approximately 0.275 and for 1.2ng/ml, approximately 0.106.
Stabilization Error was calculated as the relative change in GOI expression
as the copy number increases from a minimum copy number (Cmin) to infinity
(approximately equal to the expression level at the copy number of
pUC19-derived pMB1. This may point towards the ability of CRISPR stabilized
promoters to be tuned to the desired stabilization error-relative strength
level by changing the expression level of dCas9.
Lower expression levels at higher dCas9 expression levels may be explained
as a result of growth rate reduction and cell morphology changes from dCas9
overexpression.

### Riboswitches

Aiming to incorporate translational control to our promoters we introduced
two Theophylline responsive Riboswitches which we thoroughly characterized.
As a reporter, we chose sfGFP fused with the first 99 nucleotides of
luciferase to avoid possible changes in the secondary structure of the
aptamer caused by sfGFP.
The riboswitches’ characterization devices, [BBa\_K2839002](http://parts.igem.org/Part%3ABBa_K2839002),
[BBa\_K2839006](http://parts.igem.org/Part%3ABBa_K2839006),
were inserted into pSB1C3 vector. For the sfGFP fluorescence intensity
measurements we used flow cytometry.

Figure 6. Theo27 response to differential induction by theophylline.
Fluorescence was measured 7 hours after initial induction with
theophylline.

The results prove that both riboswitches reacted to theophylline,
facilitating inducible expression of sfGFP. Specifically, sfGFP activity
increased in direct proportion to the concentration of the ligand.

Figure 7. Behaviour of 12.1 Theophylline riboswitch driving the
expression of the same marker with different N-terminus amino-acid
sequences.

12.1 Riboswitch does not respond to theophylline when driving sfGFP
expression, but retains functionality upon fusion of the sfGFP with the
first 99 nucleotides of the luciferase protein, after the start codon.

FIgure 8. TALE stabilized promoter with translational control.
Induction of the TALE stabilized promoter driving sfGFP expression
under the regulation of the 12.1 theophylline riboswitch. (n=3).

The proper function of the 12.1 riboswitch under TALE promoter
stabilization proves that the two systems, are, as expected (link design),
orthogonal to each other. The drop in expression at 8mM theophylline, can
be credited to the negative effects on growth that high theophylline
concentrations (>5mM) exhibit.

From figures 7 and 8, it is evident that the 12.1 theophylline riboswitch
driving sfGFP fusion protein, displays better activation ratio than Theo27
theophylline responsive riboswitch. Therefore, we incorporated it
downstream of the **TALEsp1-Pupsp1 stabilized promoter, achieving on the
fly
inducibility**.

### Conclusions

* **We characterized two TALE stabilized promoter variants, and
  proved their expected function.**
* **We designed and successfully implemented a CRISPRi stabilized
  promoter and confirmed that CRISPRi has the potential to
  stabilize promoters over a wide range of plasmid copy numbers
  for different dCas9 expression levels.**
* **Stabilized promoters do not interfere with the function of
  downstream riboswitches.**
* **Successfully controlled stabilized gene expression with
  theophylline responsive aptamer.**
* **Restoration of functionality of a non functional part when
  driving a common reporter (sfGFP), by fusing it with the 99
  nucleotides of a different one.**
* **We characterized 3 riboswitches.**

### What we unfortunately did not achieve

* **Even though we successfully cloned a heterologous AND gate and
  its stabilized variant into different plasmid backbones, we
  didn’t manage to properly characterize their functionality and
  behaviour over a range of different copy numbers.**
* **We didn’t succeed in plotting a CRISPRisp response function to
  determine the cooperativity of repression and gain further
  insight into the behaviour of this system.**
* **We did not manage to characterize a RNA-based iFFL stabilized
  promoter utilizing a transcriptional attenuator, due to time
  constraints.**

### Future Experiments

* **Properly stabilize and prove the improvement of the heterologous
  AND-gate.**
* **Better characterize CRISPRi stabilized promoters over more copy
  numbers and in different E. coli strains.**
* **Achieve promoter stabilization via the use of a transcriptional
  attenuator.**