Revision as of 08:43, 16 September 2015

Project Results

Transformation Efficiency Kit, RFP construct (iGEM)

Before using our competent E. coli TOP10 cells in the important experiments, we used the Transformation Efficiency Kit to test the efficiency of our competent cells!

The kit includes five vials of each different DNA concentration: 50pg/μl, 20pg/μl, 10pg/μl, 5pg/μl, 0.5pg/μl of purified DNA from BBa_J04450 (RFP construct) in plasmid backbone pSB1C3.

The first test transformation (50 μl of competent cells, 20 μl plated) showed very poor results:

Results of first test transformation with own competent E.coli TOP10 by using Transformation Efficiency Kit, RFP construct (iGEM)

DNA concentration	0.5pg/μl	5pg/μl	10pg/μl	20pg/μl	50pg/μl
# of colonies	0	0	2	0	13
efficiency (cfu)	0	0	3.8x10⁶	0	3.4x10⁶

Results	efficiency
average	1.4x10⁶
weighted	3.5x10⁶

The second test transformation (200 μl of competent cells, 100 μl plated) showed still poor results but we decided to continue working with our cells:

DNA concentration	0.5pg/μl	5pg/μl	10pg/μl	20pg/μl	50pg/μl
# of colonies	1	13	1	35	28
efficiency (cfu)	8.0x10⁶	1.0x10⁷	4.0x10⁵	7.0x10⁶	2.3x10⁶

Results	efficiency
average	5.6x10⁶
weighted	3.7x10⁶

RFP construct

RFP CONSTRUCT

RFP (RFP DsRed) was amplified from by PCR (Fig.1). Colonies on a plate were given to us by the Applied and Genomic Microbiology department. Primers contained restriction sites for KpnI and SacI in order to make them compatible for insertion into the multiple cloning site of the pBAD A vector.

After purification of the PCR product it was ligated into pJET1.2 by subcloning (blunt end ligation). This vector serves to clone the fluorescent protein without triggering its activity, which may interact with the expression vector or the chosen E.coli strain.

Ligation into pJET1.2 was followed according to the protocol in the methods collection. After over-night incubation colonies were picked, plasmids extracted with the QIAGEN QIAprep Spin Miniprep Kit and restricted with KpnI and SacI (Fig.2).

Once restriction controls showed the correct bands, both pJET_RFP_3 and pJET_RFP_7 were sent for sequencing by the G2L laboratory.

RESULT

Both sequences were correct. We decided to work with pJET_RFP_3. The plasmid was transformed with E.coli TOP10 and E.coli BL21. Cryocultures were frozen for a strain collection.

Afterwards the RFP_3 insert was ligated into pBAD A via the T4 ligase system according to the protocol in the methods collection and transformed into E.coli TOP10.

MICROSCOPY

To check the red fluorescence transformed E.coli TOP10 were examined by fluorescence microscopy with an excitation by 536 nm. RFP DsRed should show an emission by 582 nm.

Unfortunately no fluorescence could be detected throughout the whole project. Nevertheless the DNA sequence of the construct is correct. It might be a problem of expression.

Esterase and Phosphatase construct

ESTERASE AND PHOSPHATASE CONSTRUCTS

Esterase was amplified from pET101_E064 and phosphatase from pTOPOXL_PLP07 by PCR for each enzyme (Fig.1). Both plasmids were given to us by the Applied and Genomic Microbiology department. Primers contained restriction sites KpnI and SacI for esterase and XhoI and PstI for the phosphatase in order to make them compatible for insertion into the multiple cloning site of the pBAD vector .The genes for these two enzymes had been found in screenings of metagenomic libraries.

After purification of the PCR products, they were ligated into pJET by blunt end ligation. This vector serves to clone the enzymes without triggering their activity, which may interact with the vector or the E.coli. In the case of phosphatase, the PCR products needed to be purified by gel extraction, to eliminate left over template plasmids, which could interfere with the transformation.

Ligation into pJET was followed according to the protocol in the methods collection. After over-night incubation colonies were picked, plasmids extracted with a Quiaprep spin Miniprep kit and restricted with either KpnI and SacI or XhoI and PstI, depending on the Enzyme (Fig.2&3).

Once restrictions showed the correct bands, both pJET containing esterase and phosphatase were sent for sequencing by the G2L laboratory.

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 <p>Esterase was amplified from pET101_E064 and phosphatase from pTOPOXL_PLP07 by PCR for each enzyme (Fig.1). Both plasmids were given to us by the Applied and Genomic Microbiology department. Primers contained restriction sites <em>Kpn</em>I and <em>Sac</em>I for esterase and XhoI and PstI for the phosphatase in order to make them compatible for insertion into the multiple cloning site of the pBAD vector .The genes for these two enzymes had been found in screenings of metagenomic libraries.</p>
 <img src=" https://static.igem.org/mediawiki/2015/1/15/PCR_esterase_phosphatase_iGEM_Team_Goettingen_2015.jpeg

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