Revision as of 17:00, 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:

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.

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 and restricted with KpnI and SacI (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 dockerin. Both components (restricted with KpnI and SacI) were ligated by the T4 ligation system. After over-night incubation colonies were picked, plasmids extracted with the QIAGEN QIAprep Spin Miniprep Kit and restricted with EcoRI (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 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 the pBAD_RFP_3, nor in the pBAD_RFP_ACEL construct. 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

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@@ Line 284: / Line 284: @@
 <html>
-<p>Both sequences were correct. We decided to work with pJET_RFP_3. The plasmid was transformed with <em>E.coli</em> TOP10 and <em>E.coli</em> BL21. Cryocultures were frozen for a strain collection.</p>
+<p>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 <em>E.coli</em> TOP10 and <em>E.coli</em> BL21. Cryocultures were frozen for a strain collection.</p>
-<p>Furthermore the RFP_3 insert was ligated into pBAD A by the T4 ligation system (sticky end ligation) according to the protocol in the methods collection and transformed into <em>E.coli</em> TOP10. This vector serves to express the fluorescent protein by triggering its activity.</p> After over-night incubation colonies were picked, plasmids extracted with the QIAGEN QIAprep Spin Miniprep Kit and restricted with <em>Kpn</em>I and <em>Sac</em>I.</p>
+<p>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 (<em>Kpn</em>I and <em>Sac</em>I). 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 <em>E.coli</em> TOP10. This vector serves to express the fluorescent protein by triggering its activity.</p> After over-night incubation colonies were picked, plasmids extracted with the QIAGEN QIAprep Spin Miniprep Kit and restricted with <em>Kpn</em>I and <em>Sac</em>I.</p>
 <p>To build a component for our Flexosome, we decided to fuse pBAD_RFP with the ACEL dockerin. Both components (restricted with <em>Kpn</em>I and <em>Sac</em>I) were ligated by the T4 ligation system. After over-night incubation colonies were picked, plasmids extracted with the QIAGEN QIAprep Spin Miniprep Kit and restricted with <em>Eco</em>RI (Fig.3).</p>

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