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	====Notebook====		====Notebook====
-	===Idea Project===	+
	==='''May 24'''===		==='''May 24'''===

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Revision as of 13:47, 11 October 2010

Contents 1 Notebook 2 May 24 3 July 30 4 July 29 5 July 28 6 July 27 7 July 26 8 July 23 9 Meeting July 21 10 July 20 11 Meeting June 26 12 Wetlab 103: Let's make an E. coli that fluoresce (or some simple BioBrick project) 13 Wetlab 102: Let's raise a few E. coli 14 Wetlab 101: Tools of the lab and their use 15 iGEM workshop follow up: Safety, ethics 16 iGEM workshop follow up: Software tools 17 DNA 104: BioBricks Protocol 18 INDICASAT lecture: Innovation 19 INDICASAT lecture: Drug discovery in nature 20 DNA 103: DNA modification, plasmids 21 DNA 102: Protein creation relationship to cellular function. 26 May 22 DNA 101: Information store, replication. 26 May

Notebook

May 24

Development of a genetically engineered E. coli that can produces the rhamnosyltranferase 1, for the production of a biosurfactant.

Steps to follow:

1.Find the gene sequences in pubmed.

2.In which bacteria is found.

3.After we found the gene, we designed two different set of primers.

4.Look for the sequences of the restriction enzymes E, X, P and S, inside the gene sequence.

5.If we found in the gene sequence a recognition sequence for the restriction enzymes.

6. So we should look for a mutagenesis protocol or kit.

7.We choose the Stratagene lightning mutagenesis kit. We also design three different set of primers, because the base pairs that surround the PstI unwanted sequence were different.

We found a paper which describes the Rhab1, the one we need for our project. This gene is found in Pseudomonas aureginosa We looked for the gene sequence en GeneBank, and this it is:

Rhla (Gene ID: 878955) LOCUS NC_002516 888 bp DNA

Rhlb (Gene ID: 878954) LOCUS NC_002516 1281 bp DNA

Well we found the gene; we analysed and look for the sequence of the restriction sites of E, X, P and S. And sadly we found the sequence of the PstI. That definitely is a setback, but thanks to mutagenesis, we can solve this problem. We design a set of primers, for the amplification of the Rhabl gene. For mutagenesis of our gene we choose the Stratagene lightning mutagenesis kit, and we also design another set of primers. Before this we analysed the gene and check the reading frame and verify which base pair we can be change without changing de amino acids sequence. Here it is the gene sequences, in red the codons we need to mutate.

July 30

We performed the terminator miniprep.

July 29

We again performed the E. coli transformation, with three BioBrick parts.

Electrophoresis

70mL of TBE 1X

0,7g of agarose

In each well, we added 2ul of loading buffer and 5ul of the sample. Final volume 7ul.

July 28

Electrophoresis of the digested parts:

4% 3:1 Agarose (Amresco, Cat no: 9012-36-6)

100 TBE 1X.

4g of agarose

Ethidium bromide Agarose gel: In each well we added 20 ul of sample, plus 2ul of loading buffer. Total volume=22ul.

Results: We could visualize the biobrick parts.

Transformation of E. coli using the part terminator, for which we followed the BioBrick protocol.

July 27

Miniprep Today we performed the miniprep of the E. coli transformed with the RBS and Promoter parts. This miniprep was made in triplicate.

Electrophoresis After the miniprep we made an electrophoresis in 1% agarose gel. Also, we measured the DNA concentration with a spectrophotometer:

[RBS]=30,4 ηg/μl

Digestion After the DNA concentration measurement, we performed the digestion of the RBS and Promoter parts.

Digestion mix for the Promoter:

NEBuffer2=5μl

BSA=0,5 μl

EcoRI=1 μl

SpeI=1 μl

DNA=20 μl

H2O=22,5μl

Digestion mix for the RBS:

NEBuffer2=5μl

BSA=0,5 μl

XbaI=1 μl

PstI=1 μl

DNA=20 μl

H2O=22,5μl

July 26

We performed miniprep, for the E. coli transformed with the RBS, GFP and the promoter plasmid. And then we performed an extraction of the plasmid DNA. DNA concentration was measured with the spectrometer. The measurements were performed with 260nm and 280nm, we obtained:

[RBS]=55,7 ηg/μl

[Promoter]= 7,18 μg/ml

For the analysis of the plasmid size, we prepared a 1% agarose gel.

Plasmids expected size:

RBS: psB1A2=2058bp, insert= 34bp; 2092bp

GFP: psB1A2=2058bp, insert= 742bp; 2800bp

Promoter: psB1A2=2058bp, insert= 45bp; 2103bp

We couldn’t visualize the expected bands, probably because in the first miniprep, we did not obtain enough DNA concentration. For this reason, we incubated more of the transformed E. coli in LB broth for 16h at 37°C.

July 23

We made streak plates, in LB ampi plates with the transformed bacteria. After the bacteria grew in the plates, were inoculated in LB broth. The incubation time was 48h.

Meeting July 21

The transformed bacteria grew, as expected. We picked one colony for the different parts and inoculated them in LB broth for 16 hours.

July 20

Activities:

1.Locate the BioBrick parts that we are going to assembly, in the distribution plates.

RBS: 2M, GFP: 14K, Terminator: 13D & Promoter: 15N

For the extraction we follow the protocol xxx

2.Clone the chosen BioBrick parts in competent E. coli.

We transformed 4 different bacteria, for the different BioBrick plasmids & prepared LB-ampi.

August 5

DNA concentration measurements

1.Promoter: 27,8 ng/ul

2.RBS: 42 ng/ul

3.Reporter: 40,3 ng/ul

Digestion mix Promoter:

 Buffer 2= 5  BSA=0, 5  Eco RI= 1  Spe I= 1  DNA= 17,98  Water=24, 52

Digestion mix RBS:

 Buffer 2= 5  BSA=0, 5  XbaI= 1  PstI= 1  DNA= 11,9  Water=30,6

3 hours incubation at 37ºC, in the thermocycler.

Electrophoresis of the terminator BioBrick.

We prepared 1% of agarose. In each well we add: 5ul of sample, 2 ul of loading buffer.

August 10

BioBrick Promoter electrophoresis

August 11

August 12

Plasmidic DNA extraction from RBS and promoter.

DNA concentration measurement

Meeting June 26

At this moment we have two ideas to develop. We are debating which idea is more feasible. Idea #1: Carlos´s team idea We want to produce Rhamnolipid in bacteria (E.coli) to use it as a biosurfactant. We have to see how we can isolate the rhamnolipid and be sure that the translation is functional.

Ernesto's Idea

Steps to follow:

1.Amplified the Rhamnosyltransferase 1 complex from Pseudomona auruginosa and clone this fragment. Size aprox 2.2 Kb. 2.Ligation / Transformation 3.Expression with reporter gen. 4.Isolation 5.Is the protein functional? 6.Make sure that the rhamnolipid is produced in prescence of rhamnose and fatty acid.

Notes:

We can see the reaction (enzymatic measure) by spectrometry. See kind of ligation.

But first, before the amplification we need to design our primers.

Primers design:

1.Check the gene sequence (In GenBank, FASTA format) 2.Design the primer sequence. 3.See kind of cloning. 4.M13 tail for cleavage site. 5.Look for cleavage site inside the rhamnosyltransferase 1 gene for our restriction enzymes. We can´t cut our gene in the process.

In summary the steps that we need to follow are:

I.Primer design II.PCR or amplification III.Cloning IV.Expression V.Sequencing

The most important steps are I and II. We need a good primers design and PCR if we want to have successful.

Idea #2 Ernesto team´s idea

Production of Cecropin compound. We want to produce the antibacterial cecropin compound. Naturally it is produced by insects and plants.

We want to use Anopheles gambiae, cecropin precursor. The gene is about 550 pb.

The steps to follow are the same in both groups. Both groups have to design the primers, and have all the experimental design for this week.

1.The sequence of the interested gene. 2.Design the primers. (50-100pb more at the beginning and end of the sequence). 3.Check for cloning sites. Which plasmid we are going to use, identify the restriction enzymes. 4.Assemble the blocks. In paper assemble all the system. Promoter + Ribosomal binding site + interest gene + reporter gene + translation end site.

All the design has to be on paper to analyze them and decided which idea is going to be the elected one.

Wetlab 103: Let's make an E. coli that fluoresce (or some simple BioBrick project)

• Instructor: INDICASAT, Dr. Carmenza Spadafora
• Date: (17h00-20h00 11th of June)
• Venue: INDICASAT
• Description: We identify a simple project based on past iGEM work and do our first BioBrick protocol project. Nothing innovative, but an opportunity to practice the protocols.

INDICASAT	INDICASAT	INDICASAT	INDICASAT
INDICASAT	INDICASAT	INDICASAT	INDICASAT
INDICASAT	INDICASAT	INDICASAT	INDICASAT

Wetlab 102: Let's raise a few E. coli

• Instructor: Lorena Coronado and and Dr. Carmenza Spadafora
• Date: (17h00-20h00 11th of June)
• Venue: INDICASAT
• Description: How does one handle E. coli?

Wetlab 101: Tools of the lab and their use

• Instructor: Dr. Patricia Llanes
• Date: (17h00-20h00 9th June)
• Venue: INDICASAT
• Description: How to handle pipettes, clean test tubes, etc.

iGEM workshop follow up: Safety, ethics

• Instructor: Dr. Ricardo Lleonart
• Date: (17h00-20h00 9th of June)
• Venue: INDICASAT
• Description: There is definetly a "safety considerations" requirement and we should address it early.

Dr. Ricardo Lleonart	Dr. Ricardo Lleonart

iGEM workshop follow up: Software tools

• Instructor: Patrick / Sara
• Date: (17h30-20h00 Monday 7th June)
• Venue: INDICASAT
• Description: Software tools available from the workshop.

Dra. Sara Pinzon

DNA 104: BioBricks Protocol

• Instructor: Sara/Patrick
• Date: (17h30-20h00 Monday 7th June)
• Venue: INDICASAT
• Description: Introduction to the BrioBricks protocol

Dr. Patrick Nee

Dr. Patrick Nee

Dr. Patrick Nee

INDICASAT lecture: Innovation

• Instructor: Dr. Jagannatha Rao, Director of INDICASAT
• Date: (17h30-20h00 Friday 4th June)
• Venue: INDICASAT
• Description: Dr. Rao's lecture on how to innovate.

INDICASAT lecture: Drug discovery in nature

• Instructor: Dr. Sergio Martinez
• Date: (17h00-20h00 Thursday 3rd June)
• Venue: INDICASAT
• Description:Would be a brainstorming session as students start thinking about projects. It would be GREAT if we could take a molecule discovered by INDICASAT in coral/frogs/nature and put it in E. coli!...

DNA 103: DNA modification, plasmids

• Instructor: Dr. Abby Guerra
• Date: 26 May 17h00-20h00
• Venue: UTP
• Description:Introduction to long tested combinant DNA techniques, the role of plasmids in bacteria, and their use as a vector for DNA modification.

Dr. Abby Guerra

Dr. Abby Guerra

Dr. Abby Guerra

DNA 102: Protein creation relationship to cellular function. 26 May

• Instructor: Dr. Abby Guerra
• Date: 26 May 17h00-20h00
• Venue: UTP
• Description: Introduction to how DNA drives cellular functions by creating proteins

Dr. Abby Guerra

Dr. Abby Guerra

DNA 101: Information store, replication. 26 May

• Instructor: Dr. Abby Guerra
• Date: 26 May 17h00-20h00
• Venue: UTP
• Description: A basic refresher course on how DNA stores information in the cell, and how it is involved in cellular replication.

Dr. Abby Guerra

Dr. Abby Guerra