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Non-antibiotic selectable and counter-selectable markers:
Sucrose Hydrolase
Background
Sucrose hydrolase is an enzyme from Escherichia coli O157:H7 strain Sakai which is involved in sucrose utilisation (Jahreis, et al., 2002). Transforming Escherichia coli K12 strains with sucrose hydrolase allows the cells to grow with sucrose as a sole carbon source, something the untransformed K12 strain cannot do. This allows this gene to be used as a selectable marker.
Cloning
cscA cloning
The cscA gene was cloned PCR with cscA specific primers. Figure 1.
Figure 1: DNA gel of PCR amplification with primers specific for cscA . The product is around 1.4-1.5 kb which corresponds to the size of cscA gene, around 1.5 kb.
Close figure 1.
This fragment was inserted into the standard BioBrick vector pSB1C3. Figure 2.
Figure 2:DNA gel of pSBIC3-cscA ligation digested with EcoRI in order to linearise the plasmid. The band is around 3.5 kb which corresponds to the vector pSBIC3 (around 2 kb) together with the cscA gene (arounf 1.5 kb)
Close figure 2.
A promoter and a reporter gene were added in front of the cscA gene (plac-lacZ). Figure 3.
Figure 3: DNA gel of pSB1C3-plac-lacZ-cscA digested with XbaI and PstI. The clear band just above 2kb corresponds both to the size of the vector and the plac-lacZ-cscA fragment. The band around 4 kb is likely to correspond to the undigested plasmid.
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cscA selection plasmid
The cscA and pSB1C3 gene were cloned using these primers. Method. pSB1C3-cscA ligation transformants are to be checked for the success of this cloning procedure.
Forward primer: GCTA gaattcgcggccgcttctagag caccagg agttgtt atg gat
Reverse primer: CATG ctgcag cggccgc t actagt a tta tt AGCACTCGG TCACAATCGT
Figure 1: DNA gel of PCR products of pSB1C3 without chloramphenicol and cscA. One product is around 1.4 kb which corresponds to the size of cscA gene, the other is around 2.2 kb which corresponds to the pSB1C3 vector without cml resistance.
Close the primers.
Method: The purified cscA and psB1C3 PCR products were digested with NdeI and ClaI. Both products were ligated and E.coli cells transformed with the ligation.
Close the method.
Characterisation
Plates
Plates characterisation showed that cscA is a suitable selectable marker- only cells which had the gene grew on sucrose as a sole carbon sourse. The drawback of this antibiotic-free selectable marker is that more time is required for the growth of the cscA cells on sucrose (overnight at 37°C+4 days at room temperature).
Figure 2: cscA cells as well as control cells were spread on LB plate, minimal plate with sucrose, minimal plate with sucrose and minimal plate with no sugars. Neither the cscA nor the control cells grow on minimal media with no sugars and grow on LB and minimal plate with glucose. However, cscA cells are growing on minimal media with sucrose while the control cells are not.
Conclusion:
We successfully cloned the sucrose hydrolase gene and inserted it into the BioBrick vector. (BBa_K917000)
We extensively characterised the sucrose hydrolase gene on plates and in liquid cultures.
We characterised the sucrose hydrolase gene linked to the arsenic promoter (BBa_K917001)
We determined its suitability as a selectable marker.
Further plans:
To check the success of pSB1C3-cscA selection plasmid and characterise it.
Methods (expand)
Inserting gene into a biobrick vecor: Cloning a PCR product into a biobrick vector protocol on OpenWetWare (http://openwetware.org/wiki/Cfrench:bbcloning) however NEB buffers were used.
DNA gel preparation: Analysing DNA by gel electrophoresis protocol on OpanWetWare (http://openwetware.org/wiki/Cfrench:AGE) however 0.5*TAE rather than 1*TAE was used.
Colony PCR screen: Screening colonies by PCR protocol on OpenWetWare http://openwetware.org/wiki/Cfrench:PCRScreening
Transformations: Preparing and using compenent E.coli cells protocol on OpenWetWare (http://openwetware.org/wiki/Cfrench:compcellprep1)
PCR reactions : Cloning parts by PCR with Kod polymerase protocol on OpenWetWare (http://openwetware.org/wiki/Cfrench:KodPCR)
Minipreps : Plasmid DNA minipreps from Escerichia coli JM109 and similar strains protocol on OpenWetWare (http://openwetware.org/wiki/Cfrench:minipreps1)
Digests to linearise the DNA frangment/determine size of insert: Analytical restriction digests protocol on OpenWetWare (http://openwetware.org/wiki/Cfrench:restriction1)
DNA purification: Purifying a PCR product from solution protocol on OpenWetWare (http://openwetware.org/wiki/Cfrench:DNAPurification1) however 165 ul NaI, 5 ul glass beads,180 ul wash buffer and 10 ul EB were used.
DNA preparation for sequencing: 2.5 ul miniprepped DNA, 2 ul water and 1 ul forward primer ( specific for biobrick prefix) or reverse primer (specific for biobrick suffix)
Nitroreductase activity assay: Overnight liquid cultures of nitroreductase strains were centrifuged at 10000 rpm for 5 mins to pellet the cells. The cells were then resuspended in 250 ul PBS and 1 ul DTT to ensure that cellular proteins are not oxidized. The solution was sonicated 6* (10 s sonication+20 s rest). The supernatant was separated from the pellet by centrifugation and used for the NADH-dependent nitroreductase activity assay.
To assess background activity NADH (5 ul) and bacterial supernatant (5 ul) were added to 0.8 ml PBS and mixed. OD340 was measured for 1 minute. DNBA(5 ul) was added to the same cuvette to start the reaction and change in OD340 was monitored for 1 minute. DMSO(5 ul) was used a control (DNBA is dissolved in DMSO)
The protein concentration of each of the supernants was estimated by by Bradford protein assay using the Pierce reagent protocol on OpenWetWare(http://openwetware.org/wiki/Cfrench:ProteinAssay)
Close methods.
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