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Team:BCCS-Bristol/Notebook
From 2009.igem.org
BCCS-Bristol
iGEM 2009
iGEM 2009
Contents |
Parts Submitted to The Registry
Promoters
- AraC promoter (regulatory without LVA tag)
Promoter-RBS fusions
- AraC Promoter fused with RBS
Reporter-Terminator hybrids
- GFP-Terminator
Carrier Proteins
- [http://partsregistry.org/Part:BBa_K259000 FhuA(BBa_K259000)] - Iron Chelator
- [http://partsregistry.org/Part:BBa_K259001 Fiu (BBa_K259001)] - Iron Chelator
BioScaffold Linkers
- [http://partsregistry.org/Part:BBa_K259002 BpuEI-BseRI (BBa_K259002)] - BioScaffold Linker Transformer
- [http://partsregistry.org/Part:BBa_K259004 BpuEI-CspCI (BBa_K259004)] - BioScaffold Linker Transformer
Outline of Project Work
Week Zero
- Familiarising with standard lab procedures for the past week (bacterial culture growth, restriction enzyme usage, agarose gel electrophoresis).
- Will start designing some biobricks for the project today.
Week 1
Canditate Proteins for Biobricks
- Isolated 3 canditate proteins to act as carriers for our biobricks. These are FhuA,Fiu & OsmE. Started to design primers to amplify the selected genes via PCR.
- Primers designed and ordered. Waiting for their arrival to do PCR! :D
Reporters,RBS,Backbones
- Decided to use 3 reporter genes, 1 RBS, 1 High Copy plasmid backbone for now.
Reporters
*RFP(Bba_E1010) *GFP(Bba_E1040) *LacZ(Bba_I732005)
RBS
*Bba_J61100 - From Anderson Family
Plasmid Backbone
*BBa_J04450 ; pSB1A3
- Tried to extract DNA from the iGEM biobricks and transfrom into bacteria.
- Transformations do not work properly with non-commercial E.coli strain (XL-1).
- Transformations worked the 2nd time round with commercial Nova Blue E.coli Strain. DNA samples in toolkit must be of low concentrations!
- Regrew bacterial colonies to amplify DNA of reporters,RBS,backbone.
- Miniprepped the DNA of Reporters,RBS,plasmid backbone and made glycerol stocks.
- Realised that we are faced with a problem when wanting to assemble biobricks for protein fusions.
Week 2
PCR
- Primers finally arrived. Did PCR to amplify carrier genes.
- PCR worked. PCR products (3 candidate proteins)ligated onto biobrick backbones pSB1A3 and pSB1A2 (contains RBS BBa_J61100).
- The plasmid backbones with the genes of interest inserted into them were used to transform the XL1-BLUE E.coli strain (although not competent enough compared to NovaBlue cells they are much cheaper!!)
- Most transformations are successful. Used transformed colonies to prepare liquid cultures so that we can proceed with minipreping them.
In frame protein fusions
- Started working on finding an easy assembly method for in-line protein fusions.
- Developed the design for a Bioscaffold-Linker transformer family (inspired by Bioscaffolds). Should allow fusions of proteins and all RFC10 biobricks in-frame after using Bioscaffold specific restriction enzymes.
- Prepared different versions of this Bioscaffold to be ordered and tested in the lab for actual functionality.
Week3
- Ligation successful only for the OsmE and FhuA inserts on pSb1A3 backbone. Unfortunately we couldn't get any of the 3 proteins on the pSB1A2 backbone.
- Seems that overxpression of these proteins on pSB1A2 is unfavourable for the cells. Hence decided to replace the existing promoter with an inducible one of our choice such as the AraC promoter.
- Promoter removed from pSB1A2 and religated. Fiu still not able to be ligated on any of the two backbones.
- In the meantime the two parts comprising of the AraC promoter were extracted from the iGEM kit plates and used to transform NovaBlue competent cells. Also liquid cultures of the transformed cells were prepared.
- Finalised designs for the Bioscaffold-Linker biobrick and ordered from GeneART/Mr.Gene the construct.
- Finding canditate primers for sequencing the first 3 carrier biobrick proteins (fhuA/osmE/fiu).
- Started thinking of a quick and dirty in-frame fusion for testing functional carrier-reporter gene fusions.
- Found easy fusion way for in line testing. Will take out end of FhuA and scar formed after RFC10 assembly of FhuA-GFP using RE's to take out FhuA end ( including TAA TAA SCAR) and part of GFP start. Will replace lost coding sequences with PCR.
- Ordered primers for the quick-n-dirty assembly method.
Week 4
- One more try to get Fiu protein on pSB1A3 backbone. NovaBlue transformed with ligation products of pSB1A3 and Fiu.
- Grew colonies for FhuA and OsmE midi preps.
- Carried out midi preps for FhuA and OsmE ( [FhuA]=378.9 ng/ul, [OsmE]=113.2 ng/ul).
- Transformed Nova Blue with biobrick part BBa_B0014 (double terminator)
- Culture colonies of BBa_B0014 transformants to get ready for mini prep.
- PCR FhuA and GFP for assembling together a quick and dirty fusion to be ready prior to BioScaffold arrival.
- Carried out colony growth (agar & liquid) for the AraC promoter (2 parts BBa_R0080 (lacking O2 region) and BBa_R0081 (the O2 region).
- Miniprepped DNA from cultures of BBa_R0080 and R0081.
- Attempted a 2-way ligation and of the BBa_R0080 and R0081 parts. The final product should be a 2-component biobrick on a plasmid in 5'-R0081-R0080-3' direction which can be transformed into cells.
- R0081 part was purified by gel extraction after being treated by double digest. R0080 and R0081 were ligated together and used to transform NovaBlue cells.
- First attempt to make the AraC promoter from the two parts failed.
- Fiu still not being ligated on ANY backbone.
Week 5
- The double terminator BBa_B0014 was taken out of the iGEM kit plates and used to transform NovaBlue cells.
- Minipreps and restriction digests were performed for terminator cultures (B0014) to check that they have the correct insert.
- Ligations set up between R0080 (on pSB1A2) and R0081 and used to transform NovaBlue competent cells.
Alternative Assembly Method
- In this method we will use standard non-iGEM-procedures to fuse FhuA and GFP. This construct will be placed downstream of an AraC-RBS fusion and upstream of a terminator. The final assembly product will be RFC10 compliant.
- The aim is to check whether a cargo protein like GFP will be transported to vesicles by one of our carrier proteins of interest like FhuA. It should be completed more quickly than the other method(at least in principle).
- It can also be used as a back up in case the bioscaffold method does not work.
- In general a PCR-amplified GFP fragment will be inserted in a pQE31 plasmid. Then a PCR-amplified FhuA fragment will be inserted upstream of the GFP.
- The whole construct will be cut and ligated upstream of an already made AraC-RBS fusion and at the end a terminator will be inserted upstream.
- Performed restriction digests of pQE31 and GFP PCR product.
- Make liquid cultures of NovaBlue transformed with complete AraC promoter.
- PCR reactions to amplify FhuA, GFP and LacZ. GFP, FhuA and pQE31 are cut with appropriate enzymes for subsequent reactions and then purified.
- LacZ reactions didn't work. We will proceed only with GFP.
- Minipreps for colonies with Arac promoter.
- Ligations set up between:
1. AraC and RBS 2. pQE31 and GFP
Week 6
- XL1-Blue transformations with ligations 1 and 2.
- Restriction digests for FhuA and OsmE to prepare their insertion downstream of the AraC-RBS costruct. Also GFP cut to fuse it with BOO14 terminator.
- Ligation set up between GFP and B0014 terminator (on pSB1AK3) and used to transform XL1-Blue.
- Transformations with AraC-RBS and pQE31-GFP didn't work. Repeated with NovaBlue cells.
- AraC-RBS transformations worked very well in contrast with pQE31-GFP and GFP-terminator that didn't work again.
- Repeat ligations between pQE31-GFP and GFP-terminator and transform XL1-Blue.
- Minipreps and Restr. Digests for AraC-RBS constructs.
- pQE31-GFP and GFP-terminator transformations failed.
- Ligations set up:
1. pQE31+GFP 2. AraC-RBS + FhuA 3. AraC-RBS + OsmE 4. GFP-terminator
Week 7
- Transform XL1-Blue with the 4 ligations (pQE31+GFP, AraC-RBS + FhuA, AraC-RBS + OsmE, GFP-terminator).
- Transformations with AraC-RBS + FhuA/OsmE worked but NOT for pQE31-GFP and GFP-terminator.Repeat transformations for the ones that didn't worked.
- Trasformation for GFP-Terminator worked and liquid cultures were prepared.
- pQE31+GFP trtansformation didn't worked. Fresh stock of pQE31 was cut open, phosphatase treated and purified. PCR set up to amplify new stocks of GFP to be ligated to the new stock of pQE31. Ligations between pQE31 and GFP with varying ratios of plasmid vector to GFP insert were carried out.
i.e. Plasmid: Insert
1:1
1:2
1:3
1:4
1:5
- pSB1A2 plasmids containing AraC-RBS-FhuA and AraC-RBS-OsmE were extracted using minipreps and later were cut with restriction enzymes and phosphatase treated. The first part of the conventional method (with bioscaffold) is finally completed.
- Minipreps were performed for the GFP-terminator-containing cultures and the plasmid on which they are found was digested with restriction enzymes to allow the incorporation of the bioscaffold, which is the next step.
- XL1-Blues were transformed with the pQE31+GFP ligations with the varying ratios of vector to insert. Moreover the bioscaffold arrived and its DNA was used to transform XL1-Blues.
- Both of the above transformations worked, liquid cultures were set up and used to carry out minipreps.
- The 4 different bioscaffold versions were cut with restriction enzymes and isolated using the gel extraction protocol. At the end of the day ligations between the cut GFP-terminator and the 4 bioscaffold versions were set up.
Week 8
- pQE31-GFP was digested and ligations with digested and purified FhuA were performed.
- XL1-Blue competent cells were transformed with the pQE31-GFP+FhuA ligations.
- The concentration of the DNA parts that we are going to send for sequencing was determined by spectrophotometry. These parts include: AraC-RBS, AraC-RBS-FhuA/OsmE, GFP-terminator and bioscaffolds-GFP-terminator.
- Liquid cultures of the bioscaffolds-GFP-terminator were prepared and minipreps performed to isolated the plasmid DNA. Then the whole bioscaffold-GFP-terminator constructs were purified by gel extraction and ligations with the AraC-RBS-FhuA/OsmE were prepared.
- Prepare liquid cultures for pQE31-GFP-FhuA.
Promoter-RBS-GFP-terminator ligation
- This will be used to check that GFP works and also to check if the AraC promoter works by trying to control its level of activity using arabinose.
- Restriction digest and gel purify GFP-terminator part and then ligate it to the already cut AraC-RBS on pSB1A2 plasmid.
Week 9
- XL1-Blue competent cells transformed with plasmid containing AraC-RBS-GFP-Terminator insert.
- Minipreps of cultures of cells containing pQE31 with FhuA-GFP insert, restriction digests and gel electrophoresis were performed to certify that the insert of interest is there.
- FhuA-GFP insert was then cut and isolated using gel extraction and ligations were prepared with open pSB1A2 containing the AraC-RBS.
- Tranformations of XL1-Blue with pSB1A2 containing AraC-RBS-FhuA-GFP (ligations) were done. Moreover liquid cultures of cells containing the AraC-RBS-GFP-termninator insert were prepared.
