iGEM 2011 — Team DTU-Denmark

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# Team:DTU-Denmark/Notebook

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# Notebook

|  |
| --- |
| Contents   * [1 Constructed plasmids](#Constructed_plasmids)   + [1.1 PChiP/PChiP(mutated)-lacZ fusion](#PChiP.2FPChiP.28mutated.29-lacZ_fusion)   + [1.2 PTet-chiX fusion](#PTet-chiX_fusion)   + [1.3 PBad-chiXR fusion](#PBad-chiXR_fusion) * [2 Strain construction](#Strain_construction) * [3 Arabinose promoter improvement](#Arabinose_promoter_improvement) |

## Constructed plasmids

### PChiP/PChiP(mutated)-lacZ fusion

The plasmids containing this DNA fragments were obtained by the following procedure:

1. **PCR**:
   1. **ChiP:** using E. coli w3110 genomic DNA as a template and TAQ enzyme. Using upstream part of ChiP and two different downstream primers (one wild type and one with mutation). we obtained two types of ChiP: **ChiP original** and **ChiP mutated**, which has altered binding site for ChiX.
   2. **lacZ:**from BBa\_I72005 biobrick using phusion enzyme.
   3. **pSB1C3**
2. **Restriction digestions**:
   1. **ChiP original** and **ChiP mutated** with XbaI and NcoI,
   2. **lacZ** with BspHI and PstI
   3. **pSB1C3** with EcoRI and PstI

      [![](/wiki/images/8/86/DTU-chip-lacZ.png)](/File%3ADTU-chip-lacZ.png)

      [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ADTU-chip-lacZ.png "Enlarge")

      Fig.1. A linearized version of the PchiP-lacZ construct inside the pSB1C3 plasmid. Restriction sites used, as well as expected fragment sizes are indicated.
3. **Ligations**:
   1. **ChiP normal** with **lacZ** and **pSB1C3**
   2. **ChiP mutated** with **lacZ** and **pSB1C3**
4. **Transformations** to *E. coli* NM522 cells.
5. **Plating the bacterial**and grow over night in 37℃ incubator
6. **Start of a liquid culture** of transformed strain.
7. **Mini prep the plasmid, do digestion analysis:** with EcoRI, PstI, EcoRV. **(fig.1)**
8. **Transformation** to NM522, IG9 and IG302 strain.
9. **Beta-Galactosidase Assay** Pick 3 transformants of chip original and chip mutated, and grow. Make betagal assays +/- ara.

### PTet-chiX fusion

The plasmids containing this DNA fragments were obtained by the following procedure:

1. **Start liquid culture of minigenes**: chiX, tetR
2. **PCR**:pSB4k5 (or any other non cam non pSB1 pSB6 plasmid)
3. **Purification of the PCR product**
4. **Mini prep minigenes**
5. **Restriction digestions**:
   1. **chiX** with XbaI and PstI
   2. **tetR** with EcoRI and SpeI
   3. **pSB1C3** with EcoRI and PstI
   4. **pSB4k5** with EcoRI and PstI
6. **Ligations** of:**tetR** with **chiX** and **pSB1C3**
7. **Transformations** to *E. coli* NM522 cells.
8. **Plating the bacterial**and grow over night in 37℃ incubator
9. **Colony PCR**with CS149/150 on transformants. **gel analysis** **(fig.2)**
10. **Start of a liquid culture** of correct ones.
11. **Mini prep the plasmid, do digestion analysis:**with EcoRI, PstI

    [![](/wiki/images/8/8f/DTU-ChiX.png)](/File%3ADTU-ChiX.png)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ADTU-ChiX.png "Enlarge")

    Fig.2. Colony PCR of cells transformed with Ptet-chiX inside pSB1C3, CS149 and CS150 primers were used. Expected band size is 1391 bp
12. **Subclone Ptet-chiX part into pSB4k5**
13. **Transformation** to *E. coli* NM522 cells.
14. **Colony PCR** with CS149/150 on transformants.Pick 4 positive colonies and grow overnight
15. **Minipreps** the corect ones, **Transform** into IG302/pSB1C3 PchiP-lacZ
16. **Beta-Galactosidase Assay** Pick 2 transformants and grow. Make Beta-Galactosidase Assay +/- atc.

### PBad-chiXR fusion

The plasmids containing this DNA fragments were obtained by the following procedure:

1. **Start liquid culture of minigenes**: chiXR
2. **PCR**:
   1. pSB3T5 (or any other non cam non pSB1 pSB6 plasmid)
   2. pBad
   3. pSB1C3
3. **Purification of the PCR product**
4. **Mini prep minigenes**
5. **Restriction digestions**:
   1. **pBad** with SpeI and EcoRI
   2. **chiXR** with XbaI and PstI
   3. **pSB1C3** with EcoRI and PstI
   4. **pSB4k5** with EcoRI and PstI
6. **Ligations** of:**pBad** with **chiXR** and **pSB1C3**
7. **Transformations** to *E. coli* NM522 cells.
8. **Plating the bacterial** and grow over night in 37℃ incubator
9. **Start liquid culture** of transformed strain.
10. **Mini prep the plasmid, do digestion analysis:**with EcoRI, PstI **(fig.3)**

    [![](/wiki/images/d/df/DTU-ChiXR.png)](/File%3ADTU-ChiXR.png)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ADTU-ChiXR.png "Enlarge")

    Fig.3. The gel picture of the PBAD-chiXR construct. The restriction enzymes used, and the sizes are indicated on the picture. EN = EcoRI + NcoI, EB = EcoRI + BamHI
11. **Subclone PBad-chiXR part into pSB3T5**
12. **Transformation** to *E. coli* NM522 cells.
13. **Colony PCR** with CS149/150 on transformants.Pick 4 positive colonies and grow overnight
14. **Minipreps** the corect ones, **Transform** into NM522/pSB1C3PchiP-lacZ and IG09/pSB1C3PchiP-lacZ
15. **Beta-Galactosidase Assay** Pick 2 transformants and grow. Make betagal assays +/- ara.

## Strain construction

The aim of this experimental section was to create strain with following gene deletetions:*lacZYA* and *chbBCARG* operon.

1. PCR of recombineering linear pieces of DNA using pSLD31 plasmid and RS233/RS234 primers pair (*lacZYA* deletion) and chb upstream/downstream primers pair. pSLD31, was provided by our supervisor Sebastién Lemire, and it contains a kanamycin resistance gene flanked by *lox66* and *lox71* sites. The upstream/downstream primers pair anneal at the *lox* sites and they contain overhangs providing homology region to the sequences flanking "gene-to-be" deleted.
2. Transformation of the *E. coli* W3110 strain containing pSLD18 plasmid (provides Red recombineering system) with pfhc3220 plasmid (containig Cre recombinase).
3. Once the strain containing both plasmids was constructed we were ready to proceed with the first round of recombineering. We followed the protocol describing this procedure to achieve first gene deletion - *$\Delta$chbBCARG*. First we screened for the gain of kanamycin resistance and did the colony PCR to confirm correct insert size. Then the Cre recombinase was activated and facilitated pop-out event of the resistance marker. We screened for loss of resistance and, again, confirmed the correct sizes with colony PCR. Colony PCRs were done using CSchb upstream/downstream primers pair. Correct strain was saved as IG7 and stored under -80oC.
4. As the next step, the IG7 strain was transformed with linear piece of DNA designed for *lacZYA* deletion. Gain of kanamycin resistance was screened for and the expected size of the insert was confirmed with colony PCR. After that the loss of resistance was conducted by growing the cells in minimum medium arabinose (0.15%). We screened for the pop-out event and colony PCRed the strain to confirm the correct recombineering. Primers used were called CSlac upstream/downstream. Finally, the strain was named IG9 and saved under -80oC.

Primers used:

* RS233 ACCAGTACGTTTTCCGCAGCTGTGGGTCAAAGAGGCATGACCTCCTTAGGAATTCTACCG
* RS234 GCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGACTAGCGGATCCTACCGTT
* chb upstream GGCCTGAGTTCTTAATTATCTTCGCGAATTATTTGCCCGATGTGTAGGCTGGAGCTGCTT
* chb downstream CCGGATGCAAGGTTCACGCCGCATCTGGCAAACATCCTCACATATGAATATCCTCCTTAG
* CSchb upstream TATCCGGCCTACAAAACTGTG
* CSchb downstream GCTGAATACGACACTGATCCAC
* CSlac upstream CGCTACAACGGGTAGCAA
* CSlac downstream TCGTTGGGCTGATGATCATAA

## Arabinose promoter improvement

Biobrick BBa\_I746908 containing PBAD/araC promoter, BBa\_I0500, fused with GFP was the scaffold for improving and testing the promoter.

1. First, set of primers was used to PCR the pBAD promoter. 5 primers annealing to the promoter, SRS56-1, -2, -3, -4, -SPL, introduced either designed changes in the -35 and -10 region (first 4 primers) or random point mutations in the spacer region (-SPL primter). The SRS57 primer was used in pair with each of the these primers.
2. BBa\_I746908 on the pSB1A2 plasmid was digested with BamHI and KpnI, while the PCR products were cut with BglII and KpnI.
3. Plasmid digestion was SAP treated in order to lower the chance of self-religating of the plasmid during the ligation step.
4. Now, the digested PCR products and the digested plasmid was ligated and transformed.
5. Then, after purifying the plasmids, the constructs could be tested for the correct insert by digesting with KpnI and BamHI. If the correct constructs were created (PCR products inserted into the plasmid) then restriction with BamHI couldn't take place. This restriction site was lost when fused with fragment digested with BglII. As a result only one band could be observed on the gel picture.
6. Once the constructs were confirmed we analysed the GFP expression in BioLektor with 0.2% arabinose as inducer.

Primers used:

* SRS56-1 tagcagatcttacctgacgctttttatcgcaactctctaTAAtttctccatacccgtttttttgggctagctactagag
* SRS56-2 tagcagatcttacTtgacActttttatcgcaactctctactgtttctccatacccgtttttttgggctagctactagag
* SRS56-3 tagcagatcttacctgacActttttatcgcaactctctaTAAtttctccatacccgtttttttgggctagctactagag
* SRS56-4 tagcagatcttacctgacgctttttatcgcaactctctacAgtttctccatacccgtttttttgggctagctactagag
* SRS56SPL tagcagatctNNNctgacgNNNNNNNNNNNNNNNNNNtactgtNNNtccatacccgtttttttgggctagctactagag
* SRS57 ccaacccgggtaacgagcaaagcactgaacacca

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