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Team:Paris Bettencourt/Notebook/Eliminate Smell
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Paris Bettencourt 2014
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Eliminate Smell
Notebook
June
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
1) Plasmid pEC-XC99E modified: Ptrc (IPTG inducible promoter) and lacI will be removed by PCR. Primers will be oPB.003 -that includes a PstI site- and
oPB.004 -that includes a XbaI site.
| 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 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 23rd
Goal: PCR agaA gBlock with oPB.001 and oPB.005
Procedure:
We are going to PCR using the
DreamTaq polymerase protocol
the agaA gBlock with the forward primer
oPB.001
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.
We will set the parameters in the machine and run the reaction overnight.
June 24th
Goal: PCR purification of the gBlock
Procedure:
We will purify the PCR product -agaA gBlock that contains prefix-agaA-suffix- obtained on
June 23rd
following the
QIAquick PCR purification kit standard protocol
.
We will run a 1% agarose gel with the purified DNA following the
standard protocol
. Gels were made by Ihab and Antonio the 23rd of June following the same protocol.
Results
After running the gel, we exposed it in UV light. We could see the Gene Ruler, but there was no band in our sample.
We think that PCR did not work. Things to change:
- 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:
We are going to PCR using the
Fusion Polymerase
for the agaA gBlock with the forward primer
oPB.001
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.
June 25th
Goal: PCR purification of the gBlock
Procedure:
We will purify the PCR product -agaA gBlock that contains prefix-agaA-suffix- obtained on
June 24th
following the
QIAquick PCR purification kit standard protocol
.
We will run a 1% agarose gel with the purified DNA following the
standard protocol
. Gels were made by Ihab and Antonio the 23rd of June following the same protocol.
Results:
- 1st raw: Gene Ruler 100 kb + 1uL Loading Dye
- 2nd and 3rd raw: purified PCR products + 1 uL of Loading Dye per 5 uL of sample. We saw the samples did not sink properly in the hole,
so we tried using more Loading Dye (raw 2 is tube A, raw 3 is tube B)
- 4th and 5th raw: 2 uL of Loading Dye for 5uL of sample (raw 4 is tube A, raw B is tube B)
We can see our construct around the 300 bp line, so we know is the gBlock.
Me measured the DNA in the samples using the Nanodrop.
- Tube A: 100 ng/uL
- Tube B: 55 ng/uL. This is a little low. It is an unexpected result considering that we pepared both tubes at the same time and following the same
protocol.
June 26th
Goal: Obtain pPB.001 (agaA gBlock digestion + ligation)
Procedure:
We will digest the purified agaA gBlock following the Thermo Fast Digestion Protocol
for enzimes EcoRI and PstI. Then we will ligate it with the linearized vector pSB1C3. We want to obtain
pPB.001
and transform E coli.
Digestion
1) We will use tube A (100 ng/uL). We added 20 uL of water + 5 uL Buffer + 20 uL DNA + 2.5 uL EcoRI + 2.5 uL PstI. We digested longer than indicated,
around 40 minutes, as we have used these enzymes before and work better this way.
2) Then we put it at 37ºC at the Isotemp for 5 min.
3) We will repeat for PSB1C3
These tubes were called Digested gBlock and Digested PSB1C3
Purification
4) We will purify both tubes using the
QIAquick kit
.
These tubes were called Dig + Pur gBlock and Dig + Pur PSB1C3
Ligation
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.
5) We will make a ligation. We will make a 1:3 proportion for the gBlock:vector. We added 3,3 uL of the linearized vector [25 ng/mL] and
2,5 uL of the insert DNA [100 ng/mL].
Purification
6) We will purify the product using the
QIAquick kit
.
This tube was called pPB.001
Goal: Transform E coli with pPB.001
Procedure:
We will transform competent E coli that were previously made by Jake following his own protocol. We will use
pPB.001
.
We will follow this
Heat Shock protocol
.
We will plate them in LBA+Cm. We will add 250 uL of Cm for 250 mL of LBA.
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 pPB.001
Procedure:
Because the plates cultured on
June 26th
have no colonies after 12h, we have decided to repeat the heat shock transformation. The mistake might be that we are using Cloroamphenicol, and we did not recover them for enough time. We recovered them during 50 min in the thermocycler.
Today we have repeated the same
Heat Shock protocol
, but we have recovered them during 2h in the incubator. The cells used were a new stock from Jake's competent E coli. The plasmid used is
pPB.001
.
Then we will plate them in LBA+Cm
Results:
We still have no colonies in the plates.
What might not work:
- Enzimes (EcoRI and PstI) work, as Ihab and Antonio have tested them. However I am cutting a linearized plasmid, so if their efficiency is not very
high, they might not work for me.
- Ligase.
- Transformation. I doubt it
- Primers are not well designed
June 29th
Goal: PCR purification of the gBlock
Procedure:
June 30th
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:
We have found out that Antonio and
Ihab
have already tested PstI and EcoRI. They seem to work quite specifically .
Also, they haven't managed to construct the plasmid, or do the
Golden Gate assembly
. We believe this is due to the Ligase and/or the Ligation buffer.
Therefore, we will use a new enzyme and buffer, ligate and transform.
Most enzymes from last year do not seem to work. We shoud not use them and wait for the new ones.
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