Team:Paris Bettencourt/Notebook/Eliminate Smell
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+ | <h5>June 20th</h5> | ||
+ | </br> | ||
+ | <h6>Goal: Change the targeting sequence of the CRISPR/Cas system</h6> | ||
+ | </br> | ||
+ | <h6>Procedure:</h6> | ||
+ | <p> | ||
+ | 1) Preparation of the vector | ||
+ | <ul><li>The following cells were taken from glycerol stock :<li></ul> | ||
+ | </p> | ||
+ | <TABLE BORDER> | ||
+ | <TR><b><TD><b>Strain (old name)</b></TD><TD><b>Carrying the plasmid (old name)</b></TD><TD><b>Resistance</b></TD><TD><b>New name</b></b></TD></TR> | ||
+ | <TR><TD>sSP004 (DH5alpha)</TD><TD>pM.001 - pCas9</TD><TD>Chl</TD><TD>sPB.008 carrying pPB.008</TD></TR> | ||
+ | </TABLE></b> | ||
<h5>June 23rd</h5> | <h5>June 23rd</h5> | ||
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<div> | <div> | ||
<div> | <div> | ||
- | We will transform competent NEB turbo E coli made following the | + | We will transform competent NEB turbo <i>E. coli</i> made following the |
<a | <a | ||
href="https://www.evernote.com/Home.action#b=60f448df-afb5-4a4d-9310-6dea624f7ab3&st=p&n=b4939a60-0f83-446a-a111-39d63eb7589c" | href="https://www.evernote.com/Home.action#b=60f448df-afb5-4a4d-9310-6dea624f7ab3&st=p&n=b4939a60-0f83-446a-a111-39d63eb7589c" |
Revision as of 23:40, 17 October 2014
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 :
- 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
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
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 |
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: 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 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 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 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.
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
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 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.
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: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: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:
September
September 3rd
Goal:Check Ligation of agaA gBlock in pSEVA // YFP and BFP in pSEVA
Procedure:
Results
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 23rd
Goal:Obtan agaA Biobrick
Procedure:
Ligate PSB1C3 with the gBlock
Following the Thermo T4 DNA Ligase protocol. Instead of leaving the tube at 22ºC for 10 min, we left it at room temperature for 45 min.
We will use tubes PSB1C3 Dig+Pur and gBlock EP Dig+Pur
We will make a ligation. We will make a 1:3 proportion for the gBlock:vector. We added 6 uL of the PSB1C3 vector [11 ng/uL] and 6 uL of the insert DNA [5 ng/uL].
**Usually we would add 3 uL for each but we are doubling the concentration of DNA and decreasing the H20**
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 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
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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