Team:Paris Bettencourt/Notebook/Eliminate Smell
From 2014.igem.org
Line 3,395: | Line 3,395: | ||
available strain : staphylococcus capitis</p> | available strain : staphylococcus capitis</p> | ||
- | <p>I test on | + | <p>I test on it these following primers :</p> |
- | it these following primers :</p> | + | |
<p> oPB.066/oPB.067 </p><p>oPB.068/oPB.069</p><p>oPB.070/oPB.071</p><p>oPB.036/oPB.037</p> | <p> oPB.066/oPB.067 </p><p>oPB.068/oPB.069</p><p>oPB.070/oPB.071</p><p>oPB.036/oPB.037</p> |
Revision as of 02:56, 18 October 2014
![](https://static.igem.org/mediawiki/2014/e/e1/Dontsweatit.png)
Notebook
June
June 7th
Goal: Design oligos to change the targetting sequence of a CRISPR plasmid. This is performed in order to target the LacZ gene of E.coli
Procedure:
This is performed on Geneious:
- Open the LacZ gene sequence on Geneious
- Find a PAM sequence in the gene (NGG)
- Copy the 30 nucleotide prior to the PAM region, and paste it as a new sequence. Convert it to oligo
- Copy it and paste in another new sequence (you now have two times the 30 nucelotides). Convert it to oligo, and Reverse Complement one of the two sequences
- Add to the sequence which isn't reverse complemented in the 5' end : AAAC
- Add to the sequence which is reverse complemented in the 5' end : AAAAC
- The two sequences should look this way :
![](https://static.igem.org/mediawiki/2014/8/88/DontSweatItPic1PB.png)
- The name of these two oligos are oPB.008 and oPB.009
- A second set of oligos was designed to not be targeting the E.coli genome
- A 30 nucleotides sequence was generated randomly and then blasted against bacteria genomes to make sure it was not complementary to any part of the E.coli genome
June 11th
Goal: Design plasmids that express agaA construct
Procedure:
Using Geneious, we first created the agaA construct:
- Promoter BBa_J23108 from the Anderson's promoter collection
- RBS from the RBS Calculator of Salis lab specific for E coli
- BioBrick Prefix + Scar from the iGEM Parts webpage
- agaA sequence codon optimized for E coli 12 (IDT online tool). We checked with Generious that there were no restriction sites for EcoRI, SpeI, ZbaI and PstI.
- Histidine tag: 6 Histidines in C-terminal were added
- Stop codon
- BioBrick scar + BioBrick suffix from the iGEM Parts webpage
To amplify this construct, we created two oligos:
Name | Sequence (5'->3') | Notes | Binding position |
oPB.001 | TATAGAATTCGCGGCCGCTTCTAGAGTGACAGCTAGCTCAGTCCTAGG | agaA forward for BioBrick vector | 1->23 |
oPB.002-BAD PRIMER | GAAGCATCATCACCATCACCACTGATACTAGTAGCGGCCGCTGCAGTTA | agaA REVERSE for BioBrick vector- NOT REVERSED | 1272->1296 |
oPB.005 | TTAACTGCAGCGGCCGCTACTAGTATCAGTGGTGATGGTGATGATGCTTC | agaA reverse for BioBrick vector | 1272->1296 |
* We noticed the 16/06/2014 that oPB.002 was NOT reversed and we designed oPB.005
Tip: When creating oligos: we add 4 nt at the beginning and the end composed by AT, to make sure the enzyme will bind properly.
We chose two backbones for the construct:
- pSB1C3 from the iGEM Parts to create a BioBrick.
- pEC-XC99E, a E coli-C glutamicum shuttle plasmid
We designed two plasmids:
1. pPB.001: Biobrick submission of agaA expression construct
1) Plasmid pSB1C3: High copy BioBrick assembly plasmid. Constitutive expression. To use in E coli
2) agaA construct
![](https://static.igem.org/mediawiki/2014/0/09/PPB001.png)
2.pPB.002:agaA expression construct. Shuttle vector E coli-C glutamicum
oPB.003 | 5'-CTGCAGATGCAAGCTTGGCTGTTTTGGCG-3' | PstI site + agaA forward for pEC-XC99E |
oPB.004 | 5'-TCTAGACACCACCCTGAATTGACTCTCTTC-3' | XbaI site + aga reverse for pEC-XC99E |
2) agaA construct
![](https://static.igem.org/mediawiki/2014/1/16/PPB.002.png)
June 13th
Goal: Since most cloning strains contain the LacZ region that is targeted by the CRISPR/Cas system. The goal is to eliminate the chloramphenicol resistance from the strain BL21-AI (in order to have a proper cloning strain that will be used for the cloning of the CRISPR/Cas system)
Procedure:
The strain BL21-AI was transformed by regular Heat Shock protocol (with 42°C for 30 seconds) the plasmid ***. The transformations were plated on ampicilin plates at 30°C for 24h. Colonies were picked and re-streaked on chloramphenicol plates and on ampicilin plates at 30°C for 24h. After confirming that there were no colonies on the chloramphenicol plates ; colonies from the ampicilin plates were picked and re-streaked on LB plates at 37°C for 24h. Colonies were picked and re-streaked on Chloramphenicol, Ampicilin and LB at 37°C for 24h.
Results:
After the last plating :
- There was no colonies on the chloremphenicol plates suggesting that the original resistance from the strain has been eliminated
- There was no colonies on the ampicilin plates suggesting that the thermosensitive plasmid has successfully been deactivated
- There was colonies on the LB plates that could be used to make fresh glycerol stocks
June 16th
Goal: Design new reverse oligo for Biobrick construction
Procedure:
We noticed oPB.002 was NOT reverse and therefore could not be used. We created oPB.005 instead:
oPB.005 | 5'-TTAACTGCAGCGGCCGCTACTAGTATCAGTGGTGATGGTGATGATGCTTC-3' | agaA reverse for BioBrick vector |
1272->1296 |
June 20th
Goal: Change the targeting sequence of the CRISPR/Cas system
Procedure:
1) Preparation of the vector
- The following cells were taken from glycerol stock:
Strain (old name) | Carrying the plasmid (old name) | Resistance | New name |
sSP004 (DH5alpha) | pM.001 - pCas9 | Chl | sPB.008 carrying pPB.008 |
- The cells were streaked on LB plates containing the proper antibiotic (Chl or Kan)
- The cells were then incubated at 37°C for 24h
- Single colonies were put in tubes containing 5mL of LB and 5uL of antibiotics
- Tubes now containing liquid culture were incubated at 37°C with agitation at 200rpm overnight
- New glycerol stocks were made using 500uL from liquid culture, mixed with 500ul of 50% glycerol
- Cells went through regular miniprep protocol using Thermofisher kit
- The pCas9 from miniprep was digested as follow :
- 3 uL of pCas9
- 2 uL of BsaI
- 5 uL of FastDigest Buffer
- 1 uL of 100X BSA
- 84 uL of dH20
- Incubated 1h @ 37°C
- The digested product was PCR purified through regular PCR purification protocol using Qiagen kit
2) Preparation of the oligos
- 20uL of the oligos from IDT were diluted in 180uL of NF water, as a working stock
- The oligos were then phosphorylated as follow :
- 1 uL of oligo I
- 1 uL of oligo II
- 5 uL of 10X T4 Ligase Buffer
- 1 uL of T4 PNK
- 42 uL of dH20
- Incubated 1h @ 37°C
- The oligos were annealed, by adding 2.5 uL of 1M NaCl, and incubating 5min @ 95°C (slow cool down). They were then diluted 10 times with dH20
3) Ligation was performed as follows
- 2 uL of digested vector
- 2 uL of annealed oligos
- 4 uL of 10X T4 Ligase Buffer
- 2 uL of T4 Ligase
- 30 uL of dH20
- Incubated 1h @ 37°C
- Overnight at room temperature
4) Transformation was performed on the fliped-out BL21-AI strain using regular Heat Shock protocol
Results:
More than a 100 colonies were observed after 24h at 37°C.
June 23rd
Goal: PCR agaA gBlock with oPB.001 and oPB.005
Procedure:
June 24th
Goal: PCR purification of the gBlock
Procedure:
Results
- Use fusion polymerase, as it is more reliable than the DreamTaq we used. DreamTaq is more for colony PCR.
- Final concentration of 200 uL, to have more DNA
-
When PCR does not work we look at:
- Annealing temperature: we start typically at 52ºC and can go down to 50ºC.
- DMSO concentration: typically 3%. Avoids missmatching
- Extension time, according to the manufacturer
Goal:PCR agaA gBlock with oPB.001 and oPB.005
Procedure:
June 25th
Goal: Make sure the cloning was successfull by performing analytical digestion and sequencing
Procedure:
- Liquid cultures were made out of colonies from the transformation
- After 24h at 37°C, regular Miniprep protocol was followed
1) Analytical digestion was performed as follows:
- 4uL 10X Green Buffer
- 2uL of EcoRI
- 2uL of XbaI
- 6uL pCas9
- 26uL of dH20
- Incubated for 30 min at 37°C in the thermocycler
2) A primer was designed 200 nucleotides upstream the region were the sequenced was changed
- The miniprep plasmid and the primer were sent to a sequencing company
Results:
![](https://static.igem.org/mediawiki/2014/9/94/DontSweatItPic2PB.png)
After gel running, it appears that the analytical digestion produced the right band sizes. Moreover, the sequencing results were matching the expected sequence.
Goal: PCR purification of the gBlock
Procedure:
Results:
June 26th
Goal: Obtain agaA Biobrick (agaA gBlock digestion + ligation)
Procedure:
Goal: Transform E coli with agaA Biobrick
Procedure:
Results:
No colonies grew after 12h. We will leave them in the incubator some more time and repeat the Heat Shock in the meanwhile
June 27th
Goal: E coli transformation with agaA Biobrick
Procedure:
Results:
June 29th
Goal: Start culture to extract plasmid
Procedure:
June 30th
Goal: Miniprep to extract a plasmid (unfinished)
Procedure:
We started a culture on June 29th. We will make a mini prep following the Mini Prep protocol. The aim is to extract the plasmid PSB6A1 to cut it with EcoRI and PstI and see the efficiency of the cut.
Results:
July
July 1st
Goal: PCR purification of the gBlock
Procedure:
July 1st
** pEC-XC99E plasmids (Cm) from the Beilfield iGEM team arrived today. We stored them at 37ºC and Pierre-Luc will make a miniprep tomorrow **Goal: Obtain agaA Biobrick
Procedure:
Goal: Transform E coli with agaA Biobrick
Procedure:
Results:
July 3rd: Study the Strains
Goal: Make sure the studied strains EC307(+) and EC307(-) that were given by a collegue scientist have the right sequence. This was performed by colony PCR and sequencing
Procedure
- Primers were designed with Geneious, 200 nucleotides upstream and dowstream of the targeted sequence of LacZ.
- 4 colony PCRs were performed : 2 on EC307(+) colonies and 2 on EC307(-) colonies. The PCR products were sent for sequencing.
Results
The sequencing results matched the expected sequences.
July 4th
Goal: Design primers for smelling genes
Procedure:
July 2nd
Goal: Miniprep to extract pSEVA351 + Glycerol stock
Procedure:
July 4th
Goal: Design primers for smelling genes
Procedure:
Name | Sequence (5'->3') | Notes | Strain | /TR>
oPB.046 | AGATTGTAGATAGTCTCGAGTCCTCC | agaA forward for primer 1 | Corynebacterium Striatum |
oPB.047 | GTCTTCGTTGAACACGTGCTGGG | agaA reverse for primer 1 | Corynebacterium Striatum |
oPB.048 | CAAGTGGTTCCACCAGCACCC | agaA forward primer 2 | Corynebacterium Striatum |
oPB.049 | ACGCCGCCGTTAGAAACCGG | agaA reverse for primer 2 | Corynebacterium Striatum |
oPB.066 | GTAAATGCTGGTAGCTCATCTTTAAAGTTCC | ackA forward | Staphylococcus epidermidis |
oPB.067 | CGTATGCCATAGTCTTCATAGTAATGATATGG | ackA reverse | Staphylococcus epidermidis |
oPB.068 | AGAAGGACATGGCATTACGTTGGG | d-ldH forward | Staphylococcus epidermidis |
oPB.069 | AGGATATGCATCATAACCAACTACTCTCG | d-ldH reverse | Staphylococcus epidermidis |
oPB.070 | GATGGTTTCTTCTTAATTGCTGCAAACCC | l-ldH forward | Staphylococcus epidermidis |
oPB.071 | CCATTTTGATTGATAAGTGTTGGTAAGCC | l-ldH reverse | Staphylococcus epidermidis |
oPB.036 | AGTGTCACCATGTTCACCAA | ldH forward | Staphylococcus aureus |
oPB.037 | CAGTAGGTTCAAGCTACGCA | ldH reverse | Staphylococcus aureus |
July 7th
1) odor exctraction
Protocol:Samples: Jalke/Henry/Alexandre cotton pads (of the 01/07)(see sweat collection)
(1) Cut a small piece of sample from Alex (A), put it in an eppendorf tube an add 600ul of PBS.
Vortex during 60 secondes.
Spin 1 minutes at 1000rpm (to put the cotton at the bottom).
Take supernatant with a 1ml syringe
Filter it (0.2ul)
(2) Cut a small piece of sample from Alex (A), put it in an eppendorf tube an add 4000ul of water.
Vortex during 60 secondes.
Spin 1 minutes at 1000rpm (to put the cotton at the bottom).
Take supernatant with a 10ml syringe
Filter it (0.2ul)
==> Smell it: The liquid has a very subtil smell (compare with the patch which have a very strong smell) Maybe the patche was too small.
(3)Cut a big piece of sample from Alex (1/4) (A), put it in an eppendorf tube an add 4000ul of water.
Vortex during 60 secondes.
Spin 1 minutes at 1000rpm (to put the cotton at the bottom).
Take supernatant with a 10ml syringe
Filter it (0.2ul)
==> Smell it: The liquid has a very subtil smell (compare with the patch which have a very strong smell). It is very slightly stronger than the (2) but it is not enought.
Results:
Sweat filtration doesn't seems to be efficient enough to capture the smell.
July 7th: Crispr Works
Goal: Determine is the designed CRISPR/Cas systems produce the expected results regarding bacteria survival (both of the strains should survive the non targeting CRISPR, and the mutant strain should survive to the LacZ targeting CRISPR more than the wild type).
Procedure
The strains were transformed by regular heat shock protocol (with 30 seconds at 42°C), either by the non targeting CRISPR or by the LacZ targeting CRISPR. The transformations products were plated on chloramphenicol plates at 37°C for 24h.
Results
Here we report the number of colonies of counting
Non targeting CRISPR | LacZ targeting CRISPR | |
---|---|---|
Wild Type | 145 | 3 |
Mutant | 132 | 44 |
July 8th
Goal: Capture the smell (volatiles compounds) in a patch. This has to be sterile, the odour has to be strong and that can stay for 5 days at least.
Protocol: Samples: Jalke/Henry/Alexandre cotton pads (of the 01/07)(see sweat collection) We used a protocol from: "Regulation of ovulation by human pheromones" and "Olfactory influences on the human menstrual cycle" Cut a medium piece form patch (from sample A). Put 500ul of ethanol (70%) and vortex (60 secondes) put immediatly the tube at -20°c. Wait for it to be frozen (2h) Take it out of freezer, wait 2-3 mins. Then all the ethanol should be evaporated, rub the piece on a new clean pad. Wait until the ethanol smell dissapear. Smell it Let the new cotton pad overnight in a box, smell it Results The cotton pad is smelling like sweat, the smell is quite strong (7 people smelled it and recognized a strong sweat odour) but there is also a small smell of ethanol. The longer you wait, smaller is the ethanol smell. The smell completely disappear overnight.
July 10th
Goal: Capture the smell (volatiles compounds) in a patch. This has to be sterile, the odour has to be strong and that can stay for 5 days at least. Use a solvent exctraction technic found in litterature.Protocol: Samples: Jalke/Henry/Alexandre cotton pads (of the 01/07)(see sweat collection) We used a protocol from: "Regulation of ovulation by human pheromones" and "Olfactory influences on the human menstrual cycle" Cut a medium piece form patch (from sample 1/2/3(3 is a whole pad)). Put 3ml of ethanol (70%) and vortex (60 secondes) put immediatly the tube at -20°c. (I've increased the amount of ethanol to be sure that the pad is sterilised) I had a control: PAD frozen without ethanol. Wait for it to be frozen (2h) Take it out of freezer, wait 2-3 mins. (1)Then all the ethanol should be evaporated, rub the piece on a new clean pad. Wait until the ethanol smell dissapear. Smell it Let the new cotton pad overnight in a box, smell it (2)When ethanol is evaporated, put the pad in a close falcon tube (3)Then all the ethanol should be evaporated, rub the piece on a new clean pad. Wait until the ethanol smell dissapear. Smell it Let the new cotton pad overnight in a box, smell it
Results houldThe cotton pad smell too much like ethanol, we should find a perfect value for the amount of ethanol putted. Then the solvent exctraction look complicated, because it appear that the smell is going away quite fast, also it is not very good that we have to keep the pad, because when people will smell it, they will directly recognize a cotton pad.
July 10th: Crispr efficiency
Goal
In order assess the discrimination properties of the designed CRISPR/Cas system, the strains were transformed by electroporation (to have more colonies and therefore a more trustworthy counting of the colonies) in 5 replicates.
Procedure
- The strains were washed with sterile water and resuspended in 1 mL after the final washing.
- The electroporation was performed following the EC2 settings, 200uL were transformed with 2uL of dialysed miniprep.
- 2h of recover followed the transformation.
- Cells were plated on chloramphenicol plates for 24h at 37°C.
Results
Here are reported the number of colonies after counting:
WT with LacZ Crispr | Mutant with LacZ Crispr | WT with Random Crispr | Mutant with Random Crispr | |
---|---|---|---|---|
Replicate n°1 | 5 | 742 | 1845 | 1745 |
Replicate n°2 | 40 | 543 | 2398 | 2354 |
Replicate n°3 | 12 | 984 | 1549 | 1890 |
Replicate n°4 | 2 | 659 | 1740 | 1754 |
Replicate n°5 | 26 | 978 | 2176 | 1956 |
July 11th
Goal: PCR of agaA gBlock with oPB.001 and oPB.005
Procedure:
We are going to PCR using the
Fusion Polymerasefor the agaA gBlock with the forward primer
oPB.001(the good one this time!) and the reverse primer
oPB.005.These two oligos include the BioBrick prefix and suffix. The aim is to add the prefix and suffix to the agaA sequence and amplify it.
July 12th and 13th
Goal: PCR purification of the gBlock
Procedure:
Results:
-
1st raw:Ready-to-use Gene Ruler (includes Loading Dye) 100 kb (5 uL)*
-
1st raw:Ready-to-use Gene Ruler (includes Loading Dye) 100 kb (5 uL)*
Goal:Obtain agaA Biobrick
Procedure:
Goal: Transform E coli with agaA Biobrick
Procedure:
Results:
On July 13th we can see what seems a colony in one of the plates. We will see tomorrow.
On July 14th we see colonies.
![](https://static.igem.org/mediawiki/2014/0/07/Colonies_14-07.jpg)
July 14th
Goal:Culture Corynebacterium glutamicum and Corynebacterium striatum
Procedure:
We will open the flask that arrived and culture Corynebacterium in Tryptophan Soy liquid medium at 37ºC overnight. After, we will plate them and culture single colonies to make competent cells and transform them. Corynebacterium glutamicum is sPB.006 and Corynebacterium striatum is sPB.007.
Goal:Obtain agaA in pSEVA (Digest pSEVA351 + ligate with agaA gBlock)
Procedure:
July 16th
Goal: Colony PCR: Check agaA Biobrick transformation
Procedure:
Yesterday July 15th Juanma runned a colony PCR with oPB.001 and oPB.005 using the
Phusion Polymerase protocol. Today we will run a 1% agarose gel with the purified DNA following the
standard protocol.
Results:
There are no bands on the gel. This might happen with Colony PCR. To double-check, we will do a MiniPrep to extract the agaA Biobrick and run a PCR with oPB.001 and oPB.005.Goal:Make competent Corynebacterium
Procedure:
July 17th
Goal:Check transformation of E coli with the agaA Biobrick (MiniPrep + PCR + Gel)
Procedure:
Results:
Again, no bands.
Goal:Make competent Corynebacterium + Transform Corynebacterium with pSEVA
Procedure:
We will use the eppenforf protocol (https://docs.google.com/file/d/0By8yVXC0fFVRMTlDd3BxQXM2Y1U/edit)
Results:
There are a lot of colonies for Corynebacterium striatum. In both plates. We will check that there is an insert. for that, we have started a culture (July 20th) and we will PCR it tomorrow.
July 18th
Goal:Check transformation of E coli with agaA Biobrick
Procedure:
Results:
The gel is empty. We cannot even see the vector.
Goal:amplify agaA in Corynebacterium Striatum
Procedure:
500uL liquid culture of Corynebactereium striatum overnight DNA extraction with RapidWater DNA isolation kit x2 number one : 25 ng/uL number two : 28 ng/uL PCR with : oPB.046/oPB.047 (agaA primers 1) oPB.048/oPB.049 (agaA primers 2) PCR protocol for 500 nucleotides PCR products: Reagent for 1X Volume: Nuclease-free water : 35 uL 5x Phusion HF Buffer : 10 ul 10 mM dNTPs : 1 ul Forward Primer (10 uM) : 0.5 ul Reverse Primer (10 uM) : 0.5 ul Template DNA : 1 ul DMSO : 1.5 ul Phusion DNA Polymerase : 0.5 ul Thermocycler Protocol: NEB PhusionTemperature (°C) | Time | |||
---|---|---|---|---|
Start | 98 | 30 s | Melt | |
Cycle 1 | 98 | 10 s | Melt | 35 Cycles |
Cycle 2 | 50 | 30 s | Anneal | 35 Cycles |
Cycle 3 | 72 | 20 s | Extend | 35 Cycles |
Finish | 72 | 5 min | Extend | |
Store | 10 | Forever | Store |
July 20th: Crispr specificity
Goal
Mix the the two strains in order to study the limit concentration of mutant that our CRISPR/Cas system could isolate.
Procedure
At the 3rd washing step, the strains were mixed in different ratios (from 50/50 to 1 / 10 to the minus 7).
Then the mixes were transformed with the regular electroporation protocol.
Results
Here are reported the ratio blue/white colonies:
LacZ Plate1 | LacZ Plate2 | LacZ Plate3 | LacZ Plate4 | Rand Plate1 | Rand Plate2 | Rand Plate3 | Rand Plate4 | |
---|---|---|---|---|---|---|---|---|
10-7 | 0.15 | 0.16 | 0.16 | 0.23 | 5.00E-01 | 7.00E-01 | 2.00E-01 | |
10-6 | 0.69 | 0.78 | 0.7 | 0.65 | 17 | 3 | 6 | 5 |
10-5 | 0.5 | 0.59 | 0.67 | 0.7 | 700 | 90 | 100 | 10 |
10-4 | 0.5 | 0.59 | 0.67 | 0.65 | 1,354 | 342 | 456 | 320 |
10-3 | 0.6 | 1 | 0.5 | 0.7 | 4,400 | 7,000 | 2,600 | 980 |
10-2 | 1 | 1 | 1 | 1 | 14,500 | 34,000 | 10,000 | 23,000 |
10-1 | 1 | 1 | 1 | 1 | 150,000 | 130,000 | 150,000 | 120,000 |
0.5 | 1 | 1 | 1 | 1 | 540,000 | 570,000 | 480,000 | 400,000 |
July 20th
Goal: Ligate agaA Biobrick + E coli transformation
Procedure:
Results:
There were no colonies 48h after.
August
August 1st
Goal: Check E coli transformation with agaA Biobrick (miniprep + digestion)
Procedure:
Results:
There are colonies on the plate. We will culture, miniprep and digest them.
August 3rd
Goal: Transform the WT and the mutant with fluorescent plasmids in order to spot CRISPR induced mutants
Procedure:
The WT was transformed with a CFP plasmid, and the mutant with a YFP plasmid, by regular heat shock protocol (30 seconds at 42°C). Cells were recovered 1h at 37°C after transformation and plated on chloramphenicol+spectomycin plates 24h at 37°C. Plates were put under optical filters to check the fluorescence.
Results:
Colonies had the expected fluorescence according to the transformation.
August 4th
Goal: Amplify ackA, l-ldH, d-ldH and ldH in microbiome armpit samples.
DNA extraction from cotton swabs.
1st step : remove cotton from stick and immerse it in PBS.
2nd step : vortex for 20s
3rd step : follow RapidWater DNA isolation kit’s protocol
PCR on DNA extraction with :
oPB.066/oPB.067 (ackA)
oPB.068/oPB.069 (d-ldH)
oPB.070/oPB.071 (l-ldH)
oPB.036/oPB.037 (ldH)
It worked for the four genes.
![](https://static.igem.org/mediawiki/2014/5/59/Gel_1.png)
Column 1 : ladder 1 kb
Column 2 : ackA
Column 3 : d-ldH
Column 4 : l-ldH
Column 7 : ldH
August 5th
Goal: Check E coli transformation with agaA Biobrick and Corynebacterium transformation with pSEVA +
Procedure:
Results:
-
1st raw:Gene Ruler 100 kb + 1uL Loading Dye
Conclusions:
Goal: Corynebacterium striatum transformation with pSEVA
Procedure:
We will use the eppenforf protocol (https://docs.google.com/file/d/0By8yVXC0fFVRMTlDd3BxQXM2Y1U/edit)
Results:
There are a lot of colonies in the plate. We will strike them to pick single colonies and check the transformation.
Goal: Sequencing PCR product from 03/08 PCR to check if I amplified right genes
Procedure:
Remind : I amplified ackA, d-ldH, l-ldH, ldH.August6th
Goal: agaA Biobrick ligation
Procedure:
Goal: Transform E coli with agaA Biobrick
Procedure:
Results:
There are colonies in the plate. We made a sub culture to pick single colonies on August 6th. On August 7th we made a liquid culture to then miniprep it and check the transformation.
August 7th
Goal: Partially run the crispr specificty study (see July 20th) but also compare the blue/white screening and the fluorescence
Procedure:
The same protocol was used (see July 20th) but on the fluorescent strains. Blue and colorless colonies were counted, and their fluorescence was checked with an optical filter.
Results:
Here are reported the number of counted colonies regarding their color and their fluorescence.
LacZ targeting CRISPR | Non targeting CRISPR | |||||||||||||||
Colorless colonies | Blue colonies | Colorless colonies | Blue colonies | |||||||||||||
CFP | 17 | 15 | 16 | 16 | 23 | 26 | 24 | 27 | 20 | 21 | 23 | 26 | 875 | 873 | 870 | 839 |
YFP | 247 | 256 | 239 | 230 | 0 | 0 | 0 | 0 | 11 | 12 | 15 | 12 | 0 | 0 | 0 | 0 |
Goal:Obtain agaA Biobrick (agaA gBlock digestion + ligation)
Procedure:
Results:
Goal:Check transformation in Corynebacterium
Procedure:
We picked 2 single colonies and incubated them in LB at 37ºC overnight.
Goal:Glycerol stock for E coli with pSEVA
Procedure:
We added 750 uL of culture and 250 uL of Glycerol 60% and stock them in the common box at -20ºC and some other tubes at -80ºC.
August 8th, 10th and 11th
Goal:Check Corynebacterium transformation with pSEVA
Procedure:
Goal: Obtain PSB1C3 and clone it in E coli
Procedure:
DNA Kit Plate Instructions
Before you use the DNA in the Distribution Kit Plates, be sure to test the efficiency of your competent cells with the
Transformation Efficiency Kit.
To use the DNA in the Distribution Kit, follow these instructions:
Note: There is an estimated 2-3ng of DNA in each well, following this protocol, assume that you are transforming with 200-300pg/ul
-
- With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate . Do not remove the foil cover, as it could lead to cross contamination between the wells.
- Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. We recommend that you do not use TE to resuspend the dried DNA.
- Transform 1ul of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.
- Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
- Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.
* To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance.
Note: There is not enough DNA in each well to perform anything but transformations
Transformation
Results:
div> I have too many colonies and they do not look red, just redish. So two things:Goal:amplifying and sequencing smell genes from a mix of 10 people’s DNA extraction and test control.
Procedure:
1> To use like control, I chose an available strain : staphylococcus capitis
I test on it these following primers :
oPB.066/oPB.067
oPB.068/oPB.069
oPB.070/oPB.071
oPB.036/oPB.037
![](https://static.igem.org/mediawiki/2014/1/1d/Gel_2.png)
Column 1 : ladder 100bp
Column 2 : ackA
Column 3 : l-ldH
Column 4 : d-ldH
I can’t amplify d-ldH in Staphylococcus capitis.
1st step: 10 people rub cotton swab on their armpit.
2nd step: After immersing the cotton in PBS I mix all PBS together in order to get a mix of samples.
3rd step: I follow the RapidWater DNA isolation kit protocol.
DNA nanodrop
Mix : 48 ng/uL
1 : 50 ng/uL
2 : 51 ng/uL
3 : 24 ng/uL
4 : 30 ng/uL
5 : 30 ng/uL
6 : 41 ng/uL
7 : 43 ng/uL
8 : 51 ng/uL
9 : 32 ng/uL
10 : 42 ng/uL
11 : 26 ng/uL
12 : 70 ng/uL
PCR for each sample with following primers :
oPB.066/oPB.067
oPB.070/oPB.071
oPB.068/oPB.069
oPB.036/oPB.037
August 12th
Goal:agaA Biobrick with new PBS1C3
Procedure:
Results:
August 15th and 16th
Goal:Check E coli transformation with agaA Biobrick
Procedure:
Results:
We can see the pSB1C3 but not agaA. We are going to make a culture of the second plate we plated, and try again with more colonies tomorrow.
August 17th
Goal:Check E coli transformation with the agaA Biobrick
Procedure:
Results:
August 21st
Goal: Study in more details the colorless colonies having a CFP fluorescence
Procedure:
Colony PCR were performed following the protocol used June 25th. The colony were also re-streaked on X-gal.
Results:
The sequences matched the expected sequence of a WT strain. The colonies were blue on X-gal. We can deduce unperfect efficiency of the blue-white screening and re-adjust the figure on Crispr efficiency.
September
September 3rd
Goal:Check Ligation of agaA gBlock in pSEVA // YFP and BFP in pSEVA
Procedure:
Results
September 4th •Design des oligos
Goal: Design with Geneious primers for PCR cloning in order to clone the CRISPR/Cas system into the pSeva vector.
Procedure
The primer design was made following the regular protocol (see 1st of June), but were added tails containing restriction sites for XbaI and SalI.
September 8th •Cloning
Goal: Clone the whole CRISPR/Cas system in the pSeva vector in order to be able to transform a broad range of strains.
Procedure
PCR were performed following the regular protocol.
PCR products were PCR purified, following the regular Qiagen PCR purification protocol.
Purified products were digested with XbaI and SalI, following the same protocol as the one followed the 25th of June.
Digestion products were PCR purified.
Purification products were used for ligation at room temperature for 2h, following this preparation :
- 1uL of T4 Ligase
- 2uL of Ligase Buffer
- 15uL of NF water
- 1uL of vector
- 1uL of insert
The ligation products were transformed by electroporation in BL21-AI, and plated on Chloramphenicol plates for 24h at 37°C.
Results
Around 500 colonies were found after 24h hours of growing.
September 16th
Goal:Transform agaA+pSEVA in E. coli
Procedure:
**There was a previous attempt where I directly transformed Corynebacterium with agaA+pSEVA. The rate of transformation of the product of a ligation is too low. First, we have to transform E. coli, that has a higher rate. Then we miniprep this plasmid and transform it in Corynebacterium.**
Results
September 17th
Goal:Check E. coli transformation withagaA+pSEVA
Procedure:
Results
- 1st raw: Loadying Dye 1kb
- 2nd raw: sample from culture 1: we threw it away. We do not see anything!
- 3rd raw: sample from culture 2. Not the right band size
September 18th
Goal:Transform Corynebacterium striatum with agaA+pSEVA
Procedure:
We will use the eppendorf protocol (https://docs.google.com/file/d/0By8yVXC0fFVRMTlDd3BxQXM2Y1U/edit) for Corynebacterium electroporation.
Procedure:
Cells were grown for approximately 18 h at 30ºC in a rotary shaker at 200 r.p.m. in 400 ml LB supplemented with kanamycin (50 pg ml-l) and glycine (2.5%).
Goal:Make competent Corynebacterium
Procedure:
We will use the eppendorf protocol (https://docs.google.com/file/d/0By8yVXC0fFVRMTlDd3BxQXM2Y1U/edit) for making competent cells, but when growing them overnight, we will follow Haynes and Britz protocol (The effect of growth conditions of Corynebacterium glutamicum on the transformation frequency obtained by electroporation). We did this because its been proven that adding glycine to the medium overnight increases the electrotransformation efficiency. This is the different part of the protocol:
Procedure:
Cells were grown for approximately 18 h at 30ºC in a rotary shaker at 200 r.p.m. in 400 ml LB supplemented with kanamycin (50 pg ml-l) and glycine (2.5%).
September 20th •Study of clones
Goal: Study if the cloning was effective.
Procedure
- Liquid cultures were made out of colonies.
- The liquid cultures were minipreped, and the minipreps were digested with HindIII and ran on a gel.
- The minipreps were also sequenced.
Results
The expected bands appeared on the gel (around 6100, 2300, 1700).
The expected sequence resulted from the sequencing.
September 23rd
Goal:Obtan agaA Biobrick
Procedure:
Results:
We runned a 1% agarose gel. The gel shows that there is nothing on the PSB1C3 tube. We will alplify PSB1C3 from Antonio's stock using oPB.082 and oPB.083 using.
Goal:PCR PSB1C3
Procedure:
PCR of PSB1C3 from Antonio's box using the Fusion Polymerase and primers oPB.082 and oPB.083.
September 24th
Goal:Transform agaA+pSEVA in E. coli
Procedure:
We will purify the PCR product obtained on Sep 23rd following the QIAquick PCR purification kit standard protocol.
Goal: Obtain agaA Biobrick (new PSB1C3 digestion + ligation)
Procedure:
Goal:Transformation of Corynebacterium striatum with agaA +pSEVA
Procedure:
We will use the eppendorf protocol (https://docs.google.com/file/d/0By8yVXC0fFVRMTlDd3BxQXM2Y1U/edit) for Corynebacterium electroporation. As a contol, we will also transform Corynebacterium striatum with V5 from Jake (pSEVA+cerulean).
Results:
September 25th
Goal:Old agaA Biobrick analytical digestion
Procedure:
Results:
There is nothing in the gel
Goal:Old agaA Biobrick analytical digestion
Procedure:
Goal:Ligation agaA gBlock in pSEVA
Procedure:
September 26th
Goal: agaA Biobrick miniprep and analytical digestion
Procedure:
Results:
We can see two bands. One around 2kb and other around 1.5 kb. This would be PSB1C3 and the gBlock. We will make a stock of colony 4.
September 27th
Goal:agaA+pSEVA miniprep and analytical digestion
Procedure:
We will check the tube of E coli with agaA+pSEVA (4 different colonies). We will do a miniprep and analytical digestion.
Results:
We can see pSEVA band at 5000 bp and there is another band around 1500 bp. It is not a very strong band but is there. Also, the culture tube smells like body odor, so even if this band is not very strong, we think it is agaA. We will use the miniprep product of colony 1 to transform Corynebacterium striatum.
Goal:Transform Corynebacterium striatum with agaA+pSEVA
Procedure:
September 27th •Killing Efficiency
Goal: Compare the efficacy of the new CRISPR vector with the previous one.
Procedure
The same expriment as July 10th was conducted, for the pCas9 and the pSeva vectors.
Results
WT with non targeting pCas9 | Mutant with non targeting pCas9 | |
---|---|---|
Replicate 1 | 1845 | 1745 |
Replicate 2 | 2398 | 2354 |
Replicate 3 | 1549 | 1860 |
Replicate 4 | 1740 | 1754 |
Replicate 5 | 2176 | 1956 |
WT with non targeting pSeva | Mutant with non targeting pSeva | |
Replicate 1 | 2398 | 2230 |
Replicate 2 | 1876 | 1984 |
Replicate 3 | 2784 | 2093 |
Replicate 4 | 2009 | 2092 |
Replicate 5 | 1997 | 1700 |
WT with LacZ targeting pCas9 | Mutant with LacZ targeting pCas9 | |
Replicate 1 | 5 | 742 |
Replicate 2 | 40 | 543 |
Replicate 3 | 12 | 984 |
Replicate 4 | 2 | 659 |
Replicate 5 | 26 | 978 |
September 29th
The sequencing of agaA Biobrick and agaA+pSEVA was negative, so we will throw again everything and start from the very beggining
Reagent | Volume |
Plasmid | 30 ul |
H2O | 135 ul |
10x FastDigest Green Buffer | 20 ul |
FastDigest XbaI | 5 ul |
FastDigest PstI | 5 ul |
FastAP | 5 ul |
Total Volume | 200 ul |
Gel extract and purify. The vector concentration after purification was 6.5 ng/ul
![](https://static.igem.org/mediawiki/2014/c/ca/AgaA_30-09_Dontsweatit_PB.png)
Template gBlock agaA 1:50 dilution
Reagent | Volume | |
1x | 4x | |
Nuclease-free water | 71 ul | 284 ul |
5x Phusion HF Buffer | 20 ul | 80 ul |
10 mM dNTPs | 2 ul | 8 ul |
Forward Primer (10 uM) | 1 ul | 4 ul |
Reverse Primer (10 uM) | 1 ul | 4 ul |
Template DNA (Plasmid, 1:10 dilution) | 1 ul | 4 ul |
DMSO | 3 ul | 12 ul |
Phusion DNA Polymerase | 1 ul | 4 ul |
Total Volume | 100 ul |
Thermocycler Protocol: NEB Phusion | ||||
Temp | Time | |||
Start | 98 °C | 30 sec | Melt | |
Cycle 1 | 98 °C | 10 sec | Melt | 35 Cycles |
Cycle 2 | 52 °C | 30 sec | Anneal | |
Cycle 3 | 72 °C | 40 sec | Extend | |
Finish | 72 °C | 5 min | Extend | |
Store | 10 °C | Forever | Store |
PCR Product looks good
The concentration is 80 ng/ul
Digest for cloning:
Reagent | Volume |
PCR Product | 30 ul |
H2O | 135 ul |
10x FastDigest Buffer | 20 ul |
FastDigest XbaI | 10 ul |
FastDigest PstI | 10 ul |
Total Volume | 200 ul |
Resuspend in 50 ul.
The final concentration is 36 ng/ul
Vector P41 diluted 1:10 (5 ng/ul)
Insert gBlock agaA (36 ng/ul)
Enzymes: XbaI, PstI
Reagent | Volume |
Vector | 12 ul |
Insert | 5 ul |
H2O | 0 ul |
Fermentas T4 Ligase Buffer |
2 ul |
Fermentas T4 Ligase Enzyme |
1 ul |
Total Volume | 20 ul |
I ran our the ligation controls. The gel looks a little funny. I think it is overloaded. There is some evidence that the ligation worked.
Miniprep and analytical digest with XbaI, PstI. V96 is P28 (pSB1C3) + agaA I picked 6 colonies from the V96 transformation. Clones 4, 5 and 6 all produced the correct banding pattern. I chose Clone 6 because the miniprep had the highest concentraion.
We made a culture and sent it to GATC with the verification primers. The sequencing was positive.
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