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:

First test transformation of own competent E.coli TOP10 with the iGEM Transformation Efficiency Kit containing RFP

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:

Second test transformation of own competent E.coli TOP10 with the iGEM Transformation Efficiency Kit containing RFP

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

RFP

RFP (RFP DsRed) was amplified from pHT315_rfp 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/His A vector. The PCR products were examined in a 0,8% agarose gel by electrophoresis.

Fig.1) A gel of the RFP DsRed PCR product showed the bands to be at the correct height.

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, restricted with KpnI and SacI and examined in a gel (Fig.2).

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.

Sequencing of both samples was correct and proved that RFP_3 and RFP_7 were properly inserted into the pJET vector. We decided to keep on working with pJET_RFP_3. The plasmid was transformed with E.coli TOP10 and E.coli BL21. Cryocultures were frozen for a strain collection.

The next step was a big restriction of the whole plasmid extract with the correspondent pair of restriction enzymes. That allowed us to isolate the RFP inserts carrying the desired restriction sites (KpnI and SacI). The fragments were purified with the PEQLAB peqGOLD Gel Extraction Kit before T4 ligation. The RFP_3 insert was ligated into pBAD/His A by the T4 ligation system (sticky end ligation) according to the protocol in the methods collection and transformed into E.coli TOP10. This vector serves to express the fluorescent protein by triggering its activity.

After over-night incubation colonies were picked, plasmids extracted with the QIAGEN QIAprep Spin Miniprep Kit and restricted with KpnI and SacI.

To build a component for our Flexosome, we decided to fuse pBAD_RFP with the ACEL (Acetivibrio cellulolyticus) dockerin. Both components, restricted with KpnI and SacI and purified with the PEQLAB peqGOLD Gel Extraction Kit were ligated by the T4 ligation system. After transformation into E.coli TOP10 and over-night incubation colonies were picked, plasmids extracted with the QIAGEN QIAprep Spin Miniprep Kit, restricted with EcoRI and examined in a gel (Fig.3).

Fig.3) Once restriction control showed the correct band height, pBAD_RFP_ACEL was sent for sequencing by the G2L laboratory.

RESULTS

Sanger sequencing showed correct sequences for the Flexosome bit pBAD_RFP_ACEL.

MICROSCOPY

To check the activity of RFP, an induction series with 0,2%, 2% and 5% L-Arabinose in the cell culture medium was prepared. Fluorescence microscopy was performed with the RFP DsRed filter (excitation at 536 nm, emission at 582 nm). Unfortunately no fluorescence could be detected throughout the whole project with different constructs. Neither in the pJET_RFP_3, nor in the pBAD constructs (pBAD_RFP_3 and pBAD_RFP_ACEL) induced with 0,2%, 2% and 5% L-Arabinose in the medium. Nevertheless every time the DNA sequences were correct (Sanger sequencing). It might be a problem of expression.

FUTURE PLANS

In future we want to exchange our vector system (pET100 instead of pBAD) to ensure that pBAD is not somehow interfering with our chosen E. coli strain.

Esterase and Phosphatase

Phosphatase and Esterase

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).

Restriction control esterase pJET iGEM Gottingen2015.jpeg

Control restriction pJET phosphatase iGEM Goettingen2015.jpeg

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

The sequencing was successful and proved that both enzymes were properly inserted into the vector. The next step was a big restriction of the whole plasmid extract with the correspondent pair of restriction enzymes. That allowed us to isolate the enzyme inserts carrying the desired restriction sites. The fragments were purified with the peqGOLD Gel extraction kit.

The esterase was then ligated into the pBAD vector containing the CCEL (Clostridium cellulolyticum) dockering. Competent E. coli Top10 cells were transformed and the pBAD_Est_CCEL construct was isolated from the positive clones with a Quiaprep spin Miniprep kit.

A restriction control with SacI for the esterase constructs was run in a 0,8% agarose gel (Fig. 4). Samples #2, #3 and #9 correspond to the linear plasmid containing the insert. Phosphatase construct was control restricted with EcoRI (Fig. 5), in this case 13 of 14 samples showed successful insertion of the enzyme fragment into the vector.

Control restriction pBAD Est CCEL iGEM Goettingen2015.jpeg

Pho CTHE control restriction iGEM Gottingen2015.jpg

Future plans

The next steps include changing to a better expression vector in order to test and measure the activity of the enzymes, especially to ensure that the fusion with the dockerins is not interfering with the function of the enzymes. Finally the aim is to combine the purified enzyme-dockerin proteins with the synthetic scaffold and prove the self assemby of the Flexosome tool.

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