We designed total 26 primers to sub-clone all the target genes. Our genes will be cloned into pTrcHis2a (Invitrogen). Our primers have 4nt of spacer, 6nt of restriction site, ~2nt of spacer to avoid frame shifting, and 18~20nt of homology region to have total 30nt length. Primer sequences for each gene and their restriction enzymes are listed below.

We purified genomic DNA from the cultured strains; Moorella Thermoacetica (DSM60), Clostridium ljungdahlii (DSM879). All the samples were triplicated.

Results

*Note: All the concentration and purity values were measured by ‘Nano Drop 2000’ (Thermo scientific), also, will be measured by same device and settings.

Discussion

We failed to extract genomic DNA from C. ljungdahlii. It may be due to the small amount of cell. Thus we cultured C. ljungdahlii again.

We transformed pTrcHis2a empty vector into TOP10 competent cell and spread the cell onto Luria Bertani (LB) plate (1 % Ampicillin).

We amplified target genes using PCR. Total reaction volume was 50 uL. We set elongation time for about 1 kb/min.

Results

We extracted DNA from PCR products using PCR gel extraction method.

Results

We amplified target genes using PCR. Total reaction volume was 50 uL. We set elongation time for about 1 kb/min. PCR products were harvested by gel elution.

Results



Moth_2312 → PCR again

We inoculated pTrcHis2a transformants into 5 mL of LB (1% Amp).

We purified pTrcHis2a vector using plasmid DNA mini-prep.

Results


The result was not satisfactory due to the presence of undesired band. The problem seemed to be that we didn’t processed vector samples with restriction enzyme.

We amplified target genes by PCR. Total reaction volume was 50 μL. We set elongation time for about 1kb/min. PCR products were harvested by gel elution.

Results

As we failed to extract genomic DNA of C. ljungdahlii on 4/2, we cultured it again and gave it a second try.

Results

Results


PCR result was not successful. there was no band for CLJU_37610, CLJU_37630, CLJU_37640 while CLJU_37650 came up with non-specific bands.

Previously prepared genes of M. thermoacetica are treated with restriction enzymes and purified. pTrcHis2A plasmid was also double digested.

Results

Unsuccessful genes underwent PCR again.

Results


We failed to all four amplify CLJU genes by PCR.


Moth_1204 gene was harvested by gel extraction.

Genomic DNA of C. ljungdahlii was extracted, amplified, and then extracted through gel.

Results


CLJU gDNA didn’t show distinctive bands. It seemed that we failed to extract gDNA of C. ljungdahlii.

Results

Results

We tried PCR amplification of C. ljungdahlii for couple of times but none of them was successful, thus this time we changed the reaction conditions. We used two genomic DNA samples from two cultured C. ljungdahlii samples and adjusted the annealing temperature to yield better results.

Results


Here, we cannot find any PCR product, which means that the genomic DNA itself was problematic rather than PCR procedure. Thus, from this result, we could conclude that gene cloning failure is not due to wrong PCR procedure but due to problems from gDNA extraction. Or, the bacterial strain itself on which we are working may not be Clostridium ljungdahlii but something else that we cannot amplify any genes of C. ljungdahlii from this gDNA.

After several experiments, we unfortunately ran out of the stock of pTrcHis2A vector. Hence, we decided to extract it in large quantity for further use. We used NucleoBond® Xtra Midi kit.

Results

Concentration of extracted vector was 118.8 ng/µl and purity was tolerable.

We treated prepared vectors with restriction enzymes for ligation with insert genes from M. thermoacetica. Different restriction enzymes were used for each vector of different insert genes.

Results

We prepared vectors and Moth_1201, 1202, 1203, 1204, 1197, 1198, 1199, 2312, 2314 genes by treating them with restriction enzymes. Before ligation, we examined each one of them and vector’s concentration and purity. The result is as follows.

Results



Samples (insert DNA and vector) were then ligated by T4 ligase overnight.

As previously discussed, we posed a question that the reason of keep failing cloning of C. ljungdahlii gene may be due to a mistake in gDNA extraction procedure. Thus, we cultured C. ljungdahlii again and extracted its gDNA again.

Results

• Sample 1 → conc.: 11.1 ng/µl, purity: 2.08
• Sample 2 → conc.: 7.2 ng/µl, purity: 2.39

The extracted gDNA of C. ljungdahlii was too little to work on thus C. ljungdahlii has been processed through colony PCR to amplify target genes directly from the cell. To verify if gDNA extraction procedure was problematic, we used only CLJU_37610 gene primer to check if it works in the case of colony PCR.

Results


In this result, we can see some nonspecific bands but no bands on exact position of c37610 gene. It seems that vague bands shown at the bottom of the gel primers which have failed to PCR amplify the gene. From continuous trials and failures, we concluded that it is highly probable that the strain we have in stock is not actually C. ljungdahlii but something else. Current bacterial strain may have problem with antibiotics that it cannot survive during the culture step, but this has low probability since we always used proper antibiotics for every bacterial strain. Thus, we decided to rather think of substitutes of CLJU genes and their products from M. thermoacetica.

As a last trial, we performed gradient PCR with gDNA of C. ljungdahlii to yield CLJU_37610, 37630, 37640, 37650. All reaction conditions were the same as ordinary PCR except for annealing step; annealing temperature differed for each samples from 45℃ to 65℃.

Results



C. ljungdahlii genes did not appear in these gels also, telling us that gradient PCR was not successful at all. Now we strongly believe that something’s wrong with our C. ljungdahlii strain. Thus, we decided to focus on figuring out alternatives of these genes in M. thermoacetica.

We performed PCR with M. thermoacetica once more.

Results

We transformed ligated vector constructs (vectors carrying Moth_1201, 1202, 1203, 1204, 1197, 1198, 1199, 2312, 2314) into E. coli TOP10 strain.

In previous PCR result, Moth_1202, 1204, 2312 showed quite thin bands, which in other words means that there are not enough quantity of those genes. Thus, we decided to re-amplify these three genes.

Results


From this PCR process, we have failed to amplify Moth genes.

We examined each plate on which we spread transformed cells to verify which one has been successfully transformed and which one have been failed.

Results

• Successful – 1201, 1202, 1203, 2314
• Unsuccessful – 1204, 1197, 1198, 1199, 2312

To try transformation of unsuccessful genes again, we treated restriction enzyme to those genes which lacks stock of transformant vector. After digestion, we verified their length and extracted insert DNAs for ligation.

Results

• Left – digestion check
• Right – gel extraction

From agarose gel, we extracted PCR amplified Moth genes.

Results

• Left – digestion check
• Right – gel extraction

Previously extracted genes (Moth_1197, 1198) are purified in advance to ligation.

Results

• Left – digestion check
• Right – gel extraction for purification

We treated Moth_1204, 1197, 1199, 2312 genes with restriction enzymes for vector construction. After digestion, insert DNAs were ligated with counterpart vectors. These samples will be used for second trial of cloning.

Since we failed to transform five samples, Moth_1204, 1197, 1198, 1199, 2312 cloned vector, we tried again. Transformed samples were seeded and cultured until colonies of each sample are isolated.

Seeded colonies were plated afterwards and after an overnight incubation, LB agar plates with cells were examined to see if transformation was successful.

Results

Samples transformed with vectors cloned with Moth_1204, 1197, 1199 were successful while Moth_1198 and 2312 showed another unsatisfactory result. We made master plate of cells which succeeded to be transformed.

We stored transformants (Moth_1201, 1202, 1203, 2313) in -80℃ by making cell stocks of them.

We extracted vectors of successfully transformed cells to examine the sequence of inserted genes in vector and verify if the genes are those of our interest.

Results



When we first ran gel electrophoresis, we forgot to digest samples, thus we didn’t get any single bands in the picture. Right after that, after single digestion of samples, we could yield gel with single bands on proper position.

After another failure, we performed PCR again with Moth_1198, 2312 genes for another trial of transformation. After PCR amplification, we extracted and purified genes for further usage.

Results

Up to this point, we found that our PCR products did not always have satisfactory quantity and purity. Hence, we had to review and discuss over our reaction condition. Among the steps of PCR, we adjusted the temperature of annealing step to the fittest temperature for each gene by using gradient PCR method.

Results


After gradient PCR, each gene tends to show single bands of proper size.

We extracted vectors carrying Moth_1204, 1197-1, 1197-2, 1199 from transformed cells.

Results

We treated samples with restriction enzyme and Moth_1204, 1199 seemed to be successful. Thus, we planned to double digest these two and send them for sequencing.

Results

We transformed seven kinds of vectors carrying Moth_1201, 1202, 1203, 1197, 1198, 2312, 2314 except for two already successful results (Moth_1204, 1199).

For transferring vectors to recombinant strains, we first had to select a specific recombinant strain which fits our gene construct. Thus, we cultured a few kinds of recombinant strains for gene expression.

For examination of target gene expression, we had to set both positive and negative controls. As a positive control, we chose pTrcHis2/lacZ vector and transformed E. coli TOP10 strain with it. For negative control, parental pTrcHis2A vector was used.

With the seven transformed samples, (which carrying Moth_1201, 1202, 1203, 1197, 1198, 2312, 2314), we performed colony PCR to directly check the existence of inserted gene from cells without extraction of vectors. Three singular colonies were picked from LB agar plates of each sample. Before running colony PCR, we made master plate of each sample.

Results



Six samples turned out to be successfully transformed except for Moth_2312. At first, we thought it was a mistake in picking colonies from plate. However, after we failed to get any colony PCR result from 10 singular colonies, we had to admit something was seriously wrong with cloning Moth_2312 gene and figure out a solution.

Since we have two successfully transformed samples which carry Moth_1204, 1199 each, now we prepared for their expression evaluation. First we cultured cells in LB media overnight and then inoculated cell culture for induction by IPTG. The final concentration of IPTG for induction was 1mM. Induced cell cultures were sampled 0h, 3h, 6h, and 16h after induction. Harvested samples will be used for SDS PAGE later.

Other successfully transformed samples were prepared and cultured for expression evaluation. We used same concentration of IPTG again and collected samples from induced culture at the same time as the case of Moth_1204, 1199. All samples will be used for SDS PAGE.

We extracted vector from transformant cells. Vectors carrying Moth_1201, 1202, 1203, 1204-1, 1204-2, 1197, 1198, 1199, 2314 were extracted and assessed for their concentration and purity. Samples whose concentration exceeds 100 ng/μl can be used for sequencing.

Results

Our sequencing result is listed below. And detail information about sequencing is attached.

• Moth_1197 → Cloning confirmed
• Moth_1198 → Cloning confirmed
• Moth_1199 → Cloning confirmed
• Moth_1201 → Cloning confirmed
• Moth_1202 → Cloning confirmed
• Moth_1203 → Cloning faied
• Moth_1204-1 → Cloning confirmed
• Moth_1204-2 → Cloning confirmed
• Moth_2314 → Cloning confirmed

▶▶Download sequencing information in pdf format

As we failed the transformation of vector carrying Moth_2312, we started from its ligation with vector again. Then ligated product was PCR amplified and gel extracted and its concentration and purity was assessed. Samples were triplicated and one with the highest concentration and most acceptable purity will be used for further experiment.

Results

No special event!

After extraction, Moth_2312 carrying vector (pTrcHis2A) was transformed into E. coli TOP10. After transformation, we isolated colonies and examined them by colony PCR.

Results


Again, it was a total failure. We could not find a clue to solve this problem yet. Samples tend to stuck at the well and what we can see was only surplus primers.

With samples collected after IPTG induction, we performed SDS PAGE to see if inserted genes are satisfactorily expressed and produced relevant enzymes. This time we only examined soluble fraction of the protein we extracted.

Results

Moth_2312 gene was again PCR amplified and digested by restriction enzymes. Then vector was dephosphorylated and ligated with double digested Moth_2312 insert gene.

Results

Results

We tried another round of colony PCR of Moth_2312 from transformed cells. Failed again.

Results

For protein expression check, we inoculated colony two and three formed on master plate for IPTG induction.

Results

As we continuously failed to amplify Moth_2312 gene by colony PCR, this time we amplified insert DNA of Moth_2314 as a positive control.

Results



Actually, insert DNA itself was successfully amplified while Moth_2312 of transformed vector could not be amplified. After discussion, we proposed that this may be due to wrongly designed primer. In addition, we decided to do mini-prep and then try another round of PCR with cloned vector.

This time, we tried not to rely on colony PCR but rather to PCR by extracting vector. We took three samples from master plate and one colony from the original plate on which transformed cells formed colonies.

Results



No products were made and this meant that we failed to transform Moth_2312.

No special event!

We found that transformation of Moth_2312 was not successful at all thus we tried it again. This time we used two types of sample; Moth_2312 and its host vector prepared on 12/04/21 and those prepared on 12/04/27. The former is numbered #1 and the latter is numbered #2. We first purified each components of digested sample.

Results

With purified samples, we underwent ligation of them and finally transformed the vector construct into E. coli TOP10.

We performed SDS PAGE again with transformed cells. This time we examined both soluble and insoluble fractions of extracted proteins.

Results

We performed colony PCR with newly transformed cells which are supposed to be carrying Moth_2312.

Results


Again, we failed to transform Moth_2312.

In SDS PAGE result, we could not see exact result of Moth_1203 expression. Thus, we decided to transform Moth_1203 again and check it for another time. To transform Moth_2312 again, we performed PCR of both genes out of genomic DNA of M. thermoacetica.

Results


With this result, we treated Moth_1203 and its host vector with restriction enzyme, ligated them, and transformed them into E. coli TOP10 strain.

We did mini-prep of transformed samples including negative control.

Results